Channel modulators

ABSTRACT

The inventions relate to compositions and articles of manufacture comprising connexin modulators, pannexin modulators, gap junction modulators, hemichannel modulators, and pannexin channel modulators and their use, alone or in combination, in treating ocular and other disorders.

RELATED APPLICATIONS

This is a Continuation of U.S. application Ser. No. 16/673,767 filedNov. 4, 2019, which was a Continuation of U.S. application Ser. No.14/833,041 filed Aug. 21, 2015, which claimed priority to U.S.Provisional Application 62/146,128, filed Apr. 10, 2015; U.S.Provisional Application 62/147,488, filed Apr. 14, 2015; U.S.Provisional Application 62/080,217, filed Nov. 14, 2014; U.S.Provisional Application 62/085,226, filed Nov. 26, 2014; New ZealandApplication 709673, filed Jul. 2, 2015; and New Zealand Application628630, filed Aug. 22, 2014; the contents of each of which is hereinincorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Feb. 11, 2020, is namedE3697-00564_SL.txt and is 112,914 bytes in size.

FIELD

The inventions relate to connexin and pannexin channel modulators andmodulation.

TECHNICAL BACKGROUND

The following includes information that may be useful in understandingthe present invention. It is not an admission that any of theinformation, publications or documents specifically or implicitlyreferenced herein is prior art, or essential, to the presently describedor claimed inventions. All publications, patents, related applications,and other written or electronic materials mentioned or identified hereinare hereby incorporated herein by reference in their entirety. Theinformation incorporated is as much a part of the application as filedas if all of the text and other content was repeated in the application,and should be treated as part of the text and content of the applicationas filed.

Gap junctions are specialized intercellular connections that are foundbetween most animal cell-types. They are expressed in virtually alltissues of the body, with the exception of mature skeletal muscle andmobile cell types such as sperm and erythrocytes. Gap junctions directlyconnect the cytoplasm of two cells, which allows various molecules, ionsand electrical impulses to directly pass through a regulated gatebetween cells.

In contrast to occluding (tight) junctions and anchoring (adherens anddesmosome) junctions, gap junctions do not seal membranes together, noris their function to restrict the passage of material between membranes.Rather, gap junction channels permit certain molecules to shuttle fromone cell to another, thus providing physical communication channels bydirectly linking the interiors of adjacent cells.

One gap junction channel is composed of two connexons (or hemichannels),which connect across the intercellular space between adjacent cells andallow intracellular molecules to flow between those cells. Each connexonof a gap junction resides in the adjacent cell membrane and is formed bythe covalent oligomerization of six individual connexin (“Cx”, or “Cxn”)proteins. Yeager (1998) Structure of cardiac gap junction intercellularchannels, J Struct Biol 121: 231-245. Connexons can comprise one or moredifferent connexin proteins, although they are usually in the form ofhomohexamers.

The human connexin family of genes and proteins now numbers 21. (Söhl G& Willecke K. (2004). Gap junctions and the connexin protein family.Cardiovasc Res. 62: 228-232). Structural and functional diversity ofconnexin genes in the mouse and human genome, Biol Chem 383: 725-737.There is much variation in the range of connexins expressed in varioustissue types and often more than one connexin form is present within acell type. See Sohl and Willecke (2004). It is possible for variouscombinations of connexins and connexons to interact with each other,although there are compatibility restrictions. Marziano, et al. Hum MolGenet. 12:805-812 (2003) Connexin proteins and their associated gapjunction channels come in a range of sizes and configurations that arethought to offer some specificity for the chemical species that can passthrough. Niessen et al. (2000) Selective permeability of differentconnexin channels to the second messenger inositol 1,4,5-trisphosphate,J Cell Sci 113 (Pt 8): 1365-1372. All connexins share a common structurewith four transmembrane domains, two extracellular loops, a cytoplasmicloop, a short cytoplasmic amino terminus and a carboxy terminus that canvary considerably in length. Unger, et al. (1999) Electroncryo-crystallography of a recombinant cardiac gap junction channel,Novartis Found Symp 219: 22-30 & discussion 31-43. The connexin proteinsare commonly named according to their molecular weights, e.g. Cx26 isthe connexin protein of 26 kDa. The principal structural differencebetween connexin proteins is the length of the C-terminal cytoplasmictail, with connexin26 having almost no tail (16 amino acids), whileconnexins 43 and 32 have long and intermediate ones (156 and 73 aminoacids, respectively). The differences in the size and amino acidsequence of the cytoplasmic tails for different connexins has beenpredicted to be involved in the channel open and closed conformations,amongst other things. The function and/or dysfunction of gap junctionshas been implicated in a number of disorders. For example, connexin30 ismutated in Clouston syndrome (hidrotic ectodermal dysplasia), andmutations in the connexin26 gene are the most common cause of geneticdeafness. Mutations in the human connexin32 gene cause X-linkedCharcot-Marie-Tooth disease, a hereditary neuropathy, whileoculodentodigital dysplasia is generally believed to be caused by amutation in the gene that codes for connexin43.

Pannexins are a family of transmembrane channel glycoproteins thatinclude Panx1, Panx2 and Panx3. Pannexins share similar structuralfeatures with connexins, consisting of 4 transmembrane domains, 2extracellular and 1 intracellular loop, along with intracellular N- andC-terminal tails. Panx1 is expressed in many mammalian tissues, whilePanx2 and Panx3 expression is more limited. Panx1 has been linked topropagation of calcium waves, regulation of tone, mucociliary lungclearance, and taste-bud function. Panx1 is expressed in the brain,bladder, testis, and ovary, whereas Panx2 is expressed primarily in thebrain, and Panx3 is expressed in skin, cartilage, heart, kidney andcochlea. Panx1 hemichannels have been implicated in ATP release, calciumsignalling, keratinocyte and osteoblast differentiation, tastereception, cell death, post-ischemic neurodegeneration, tumoursuppression and seizure. Panx2 is involved in differentiation of neuronswhile Panx3 plays a role in the differentiation of chondrocytes,osteoblasts and the maturation and transport of sperm. Panx1 islocalized to the plasma membrane whereas Panx2 is intracellularlylocated. One major difference between connexin and pannexin channels isthat pannexin channels do not form cell-to-cell channels and it has beensuggested that the highly glycosylated extracellular loops of pannexinproteins interferes with the docking process. As with connexinhemichannels, pannexin channels are said to be activated by a number offactors, but also show some differences. Both connexin hemichannels andpannexin channels contribute to glutamate and ATP release, althoughpannexin channels are insensitive to decreases in calcium ionconcentration.

BRIEF SUMMARY

The inventions described and claimed herein have many attributes andembodiments including, but not limited to, those set forth or describedor referenced in this Brief Summary. It is not intended to beall-inclusive and the inventions described and claimed herein are notlimited to or by the features or embodiments identified in thisintroduction, which is included for purposes of illustration only andnot restriction.

It is an object of the invention to provide compounds, compositions,formulations, kits and methods for the modulation of a gap junctionchannel, hemichannel, and/or pannexin channel, and/or for the treatmentof disorders that will benefit from modulation of a gap junctionchannel, hemichannel, and/or pannexin channel. Thus, in one aspect, thepresent invention relates to methods for the modulation of a gapjunction channel, gap junction hemichannel, and/or a pannexin channel,and particularly, but not exclusively, to methods for the treatment ofdisorders for which modulation of a gap junction channel, gap junctionhemichannel, and/or a pannexin channel, may be of benefit, the methodscomprising administering a gap junction channel modulator, for example,compounds of formula I (for example tonabersat), a connexinpeptidomimetic compound, and/or one or more analogue or prodrug of anyof the foregoing compounds thereof, and/or administering a pannexinmodulator, such as a compound of formula VI (for example probenecid)and/or one or more analogue or prodrug of any of the foregoing compoundsthereof, and/or a pannexin peptidomimetic compound and/or one or moreanalogue or prodrug of any of the foregoing compounds thereof.

In one aspect, the invention provides methods for the modulation of agap junction channel and/or a hemichannel using a gap junction channelmodulator or hemichannel modulator. In some aspects, the gap junctionchannel modulator or hemichannel modulator may be a small moleculemodulator. Small molecule gap junction channel modulators include, forexample, compounds of formula I, for example tonabersat, and analoguesand/or prodrugs thereof. In some aspects, the gap junction channelmodulator or hemichannel modulator may be a peptidomimetic compound.Peptidomimetic gap junction channel modulators include, for example,peptide 5 (VDCFLSRPTEKT (SEQ ID NO: 168)) and analogues thereof. Inother aspects, the gap junction channel modulator or hemichannelmodulator may be an antisense oligonucleotide, which may be chemicallymodified or an unmodified oligonucleotide, e.g. an unmodified DNAoligonucleotide.

In some aspects this invention provides methods for the modulation of apannexin channel using a pannexin modulator. In some aspects, thepannexin modulator may be a small molecule modulator. Small moleculepannexin channel modulators include, for example, compounds of formulaVI, for example probenecid, and analogues and/or prodrugs of any of theforegoing compounds. In some aspects, the pannexin modulator may be asynthetic mimetic peptide blocker of pannexin 1. Peptidomimetic pannexinchannel modulators include, for example, ¹⁰Panx1, or an analoguethereof. In some aspects, the pannexin modulator may be a syntheticmimetic peptide blocker of pannexin 2. In some aspects, the pannexinmodulator may be a synthetic mimetic peptide blocker of pannexin 3.

In some aspects, the method comprises co-administering a gap junctionchannel modulator and a pannexin modulator. The co-administration of thegap junction channel modulator can be simultaneously with, subsequentto, or before the administration of the pannexin modulator. In someaspects, compounds of formula I, for example tonabersat, and/or peptide5, or analogues of either or both, may be co-administered with apannexin antagonist, e.g., compounds of formula VI, for exampleprobenecid, and/or one or more analogues or prodrugs of any of theforegoing compounds, and/or a synthetic mimetic peptide blocker ofpannexin 1, e.g., ¹⁰Panx1 or an analogue thereof.

In another aspect, the invention provides the use of a hemichannelmodulator in the manufacture of a medicament for modulation of a gapjunction channel and/or a hemichannel, or treatment of any of thediseases, disorders and/or conditions herein. Hemichannel modulatorsinclude, for example, compounds of formula I, for example tonabersat,and/or an analogue thereof, and/or peptide 5 or an analogue thereof,and/or an anti-sense oligonucleotide, as described herein.

In another aspect, the invention provides a gap junction channelmodulator, a hemichannel modulator, a connexin modulator, and/or apannexin modulator, such as compounds of formula I, for exampletonabersat, and/or an analogue of any of the foregoing compounds orpeptide 5 or an analogue thereof for use in the modulation of a gapjunction channel and/or a hemichannel. In one aspect, the gap junctionchannel modulator, a hemichannel modulator, a connexin modulator, and/ora pannexin modulator, preferably modulates gap junction channels and/orhemichannels and pannexins, respectively, in humans or other animals. Insome aspects the invention also features a pannexin modulator, e.g.,compounds of formula VI, for example probenecid, and/or one or moreanalogue or prodrug of any of the foregoing compounds thereof and/or asynthetic mimetic peptide blocker of a pannexin such as pannexin 1, forexample, ¹⁰Panx1 or an analogue thereof, for use in the modulation of apannexin channel. Preferred pannexin channel modulators are pannexin 1channel modulators. In some aspects, the gap junction channel modulatorand/or hemichannel modulator can be used together with a pannexinmodulator for modulation of a gap junction channel and/or a hemichanneltogether with modulation of a pannexin or pannexin channel. In someaspects, modulation of the pannexin and/or pannexin channel may bebefore, after or simultaneous with modulation of the connexin channel.In other aspects, modulation of the connexin channel may be before, orafter, modulation of the pannexin channel.

In another aspect, the invention provides a method for the treatment ofa disorder by modulation of a gap junction channel and/or a hemichannelwhere modulation of a gap junction channel and/or a hemichannel may beof benefit, the method comprising administering a connexin channelmodulator and/or a hemichannel modulator to a subject. In some aspectsthe channel modulator may be, for example, compounds of formula I, forexample tonabersat, and/or an analogue of any of the foregoing compoundsto a subject, and/or a peptidomimetic compound, such as Peptide 5 or ananalogue thereof. In some aspects where modulation of a pannexin channelmay be of benefit, the method further comprises administering a pannexinantagonist or pannexin channel modulator. Pannexin modulators include,for example, compounds of formula VI, for example probenecid, and/or ananalogue or prodrug of any of the foregoing compounds and/or a syntheticmimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1 or an analoguethereof. In some aspects, the method comprises administering compoundsof formula I, for example tonabersat, and/or an analogue of any of theforegoing compounds, together with a pannexin antagonist, e.g.,compounds of formula VI, for example probenecid, and/or one or moreanalogue or prodrug of any of the foregoing compounds and/or a syntheticmimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1 or an analoguethereof. The administration of compounds of formula I, for exampletonabersat, and/or an analogue of any of the foregoing compounds can besimultaneously, subsequently, or before the administration of thepannexin antagonist. The administration of the pannexin antagonist canbe subsequent to, or before, the administration of the connexin channelantagonist, e.g., compounds of formula I, for example tonabersat, and/oran analogue of any of the foregoing compounds. In some aspects, themethod of treatment is applied to humans. In some aspects, the method oftreatment is applied to mammals, e.g. humans.

In another aspect, the invention features the use of a pannexinmodulator in the manufacture of a medicament for the modulation of apannexin channel, or treatment of one or more of the diseases, disordersand/or conditions herein. Pannexin modulators include, for example,compounds of formula VI, for example probenecid, and/or analogues andprodruge of any of the foregoing compounds and/or a synthetic mimeticpeptide blocker of pannexin 1, for example, ¹⁰Panx1 or an analoguethereof. In some aspects, a gap junction channel modulator or ahemichannel modulator and a pannexin modulator may be use for themanufacture of a single medicament, or a pair of medicaments, for themodulation of a gap junction channel and/or a hemichannel, and themodulation of a pannexin channel.

In another aspect, the invention provides the use of a connexinmodulator such as compounds of formula I, for example tonabersat, and/oran analogue thereof or peptide 5 or an analogue thereof in themanufacture of a medicament for the treatment of a disorder wheremodulation of a gap junction channel and/or a hemichannel may be ofbenefit. In some aspects, where modulation of a pannexin channel may beof benefit, the invention features the use of a pannexin antagonist orpannexin channel modulator (e.g., compounds of formula VI, for exampleprobenecid, and/or one or more analogue or prodrug of any of theforegoing compounds and/or a synthetic mimetic peptide blocker ofpannexin 1, e.g., ¹⁰Panx1 or an analogue thereof) in the manufacture ofa medicament for the treatment of a disorder where modulation of apannexin channel may be of benefit, including for treatment of thediseases, disorders and/or conditions herein.

In some aspects, compounds of formula I, for example tonabersat, and/oran analogue of any of the foregoing compounds may be used together witha pannexin antagonist in the manufacture of a medicament for thetreatment of a disorder where modulation of a gap junction channeland/or a hemichannel, and a pannexin channel is of benefit.

In another aspect, the invention provides a gap junction channelmodulator, for example, compounds of formula I, for example tonabersat,and/or an analogue of any of the foregoing compounds or peptide 5 or ananalogue thereof or another gap junction channel modulator, for use inthe treatment of a disorder where modulation of a gap junction channeland/or a hemichannel may be of benefit. In some aspects where modulationof a pannexin channel may be of benefit, the invention also features apannexin antagonist, for example, compounds of formula VI, for exampleprobenecid, and/or one or more analogue or prodrug of any of theforegoing compounds thereof and/or a synthetic mimetic peptide blockerof a pannexin such as pannexin 1, for example, ¹⁰Panx1 or an analoguethereof, or another pannexin channel modulator, for use in the treatmentof a disorder where modulation of a pannexin channel may be of benefit,including for treatment of the diseases, disorders and/or conditionsdescribed or referenced herein. In some aspects, compounds of formula I,for example tonabersat, and/or an analogue or prodrug of any of theforegoing compounds or peptide 5 or an analogue thereof can be usedtogether with a pannexin antagonist for treatment of a disorder wheremodulation of a gap junction channel and/or a hemichannel and a pannexinchannel may be of benefit. The administration of compounds of formula I,for example tonabersat, and/or an analogue of any of the foregoingcompounds can be simultaneously, subsequently, or before theadministration of the pannexin antagonist.

In any of the aspects of this invention, any of the following may beused as the gap junction channel modulator, for example: compounds offormula I, for example tonabersat, or analogs thereof, peptide 5 or ananalogue thereof. Other compounds with some gap junction channel and/orhemichannel regulating activity include narcotics (including, forexample, isoflurane, halothane, ethane) octanol, heptanol,18α-glycyrrhetinic acid (including its metabolites), carbenoxolone,fenamates (including, for example, flufenamic or niflumic acid), cardiacglycosides (including for example, ouabain), platelet derived growthfactor (PDGF), IGF-1, carbochol, phorbol esters and arachidonic, oleicor palmitoleic acids (See Salameh A, et al., (2005) Biochim Biophys Acta1719:36-58, for further examples), and/or quinoline or mefloquinecompounds (see for example Das S et al., Biochem Biophys Res Commun(2008) 373:504-508).

In various embodiments, the gap junction channel and/or the hemichannelbeing modulated comprises one or more of connexin23, 25, 26, 30, 30.2,30.3, 31, 31.1, 31.9, 32, 36, 37, 40, 40.1, 43, 45, 46, 47, 50, 59, and62. In one embodiment, the gap junction channel and/or hemichannel beingmodulated comprises one or more of connexin 26, 30, 32, 36, 37, 40, 45and 47. In one particular embodiment, the gap junction channel and/orhemichannel being modulated comprises connexin 30 and/or connexin 43.

In various embodiments, the pannexin and/or the pannexin channel beingmodulated is one or more of pannexin 1, pannexin 2 and pannexin 3.

In another aspect, the invention provides a method for the treatment ofone or more diseases, disorders or conditions referred to herein, themethod comprising administering a gap junction channel modulator to asubject. In some aspects, the gap junction channel modulator may be, forexample, compounds of formula I, for example tonabersat, and/or ananalogue of any of the foregoing compounds or peptide 5 or an analoguethereof. In some aspects this invention also provides a method for thetreatment of one or more diseases, disorders or conditions referred toherein, the method comprising administering a pannexin modulator, aloneor in combination with one or more gap junction channel or hemichannelmodulators. In some aspects, the pannexin modulator may be, for example,compounds of formula VI, for example probenecid, and/or one or moreanalogue or prodrug of any of the foregoing compounds thereof and/or asynthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1 or ananalogue thereof. In certain embodiments, the one or more diseases,disorders or conditions is chosen from the group comprising, forexample: a chronic wound, a wound that does not heal at the/an expectedrate, a dehiscent wound; fibrosis, a fibrotic disease, disorder orcondition; abnormal or excessive scarring; a vascular disorder; tissuedamage; an orthopedic disease or disorder; inflammation or aninflammatory disease; edema.

In another aspect, the invention provides the use of a gap junctionchannel modulator or hemichannel modulator, such as peptide 5, and/or ananalogue thereof, compounds of formula I, for example tonabersat, and/oran analogue of any of the foregoing compounds, in the manufacture of amedicament for the treatment of one or more diseases, disorders andconditions referred to herein. In some aspects, the invention alsoprovides the use of a pannexin modulator, e.g., compounds of formula VI,for example probenecid, and/or one or more analogue or prodrug of any ofthe foregoing compounds thereof and/or a synthetic mimetic peptideblocker of pannexin 1, e.g., ¹⁰Panx1, or an analogue of either thereofin the manufacture of a medicament for the treatment of one or morediseases, disorders and conditions referred to herein. In some aspects,a gap junction channel modulator may be used together with a pannexinmodulator in the manufacture of separate medicaments or a combinationmedicament for the treatment of one or more diseases, disorders andconditions referred to herein. In certain embodiments, the one or moredisorder is chosen from the group comprising, for example: a chronicwound, a wound that does not heal at the/an expected rate, a dehiscentwound; fibrosis, a fibrotic disease, disorder or condition; abnormal orexcessive scarring; a vascular disorder; tissue damage; an orthopedicdisease or disorder; inflammation or an inflammatory disease; and edema.

In another aspect, the invention provides a gap junction channelmodulator or a hemichannel modulator, such as peptide 5, and/or ananalogue thereof, compounds of formula I, for example tonabersat, and/oran analogue of any of the foregoing compounds, for the treatment of oneor more diseases, disorders and conditions referred to herein. In someaspects, the invention also provides the use of a pannexin modulator,e.g., compounds of formula VI, for example probenecid, and/or one ormore analogue or prodrug of any of the foregoing compounds thereofand/or a synthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1,or an analogue of either thereof, for example, for the treatment of oneor more diseases, disorders and conditions referred to herein, eitheralone or in combination with a gap junction channel and/or hemichannelmodulator of this disclosure. In certain embodiments, the one or morediseases, disorders or conditions is chosen from the group comprising,for example: a chronic wound, a wound that does not heal at the/anexpected rate, a dehiscent wound; fibrosis, a fibrotic disease, disorderor condition; abnormal or excessive scarring; a vascular disorder;tissue damage; an orthopedic disease or disorder; inflammation or aninflammatory disease; and edema.

In another aspect, the invention provides a gap junction channel orhemichannel modulator, such as peptide 5, and/or an analogue thereof,compounds of formula I, for example tonabersat, and/or an analogue ofany of the foregoing compounds, for example, for the treatment of one ormore diseases, disorders and conditions referred to herein. In someaspects, the invention also provides the use of a pannexin modulator,e.g., compounds of formula VI, for example probenecid, and/or one ormore analogues or prodrugs of any of the foregoing compounds and/or asynthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1, or ananalogue of either thereof, for example, for the treatment of one ormore disorder referred to herein, either alone or in combination with agap junction channel modulator including those of this disclosure. Incertain embodiments, the one or more diseases, disorders or conditionsis chosen from the group comprising one or more of, for example,ischemia (including for example, perinatal ischemia, skin ischemia andcardiac ischemia), brain stroke, asphyxia, brain trauma, spinal cordinjury, heart attack, inflammatory cardiac insult (including forexample, pericarditis), reperfusion injury (including for example,cardiac reperfusion after surgery or transplant), liver reperfusionafter surgery or transplant, retinal ganglion cell (RGC) loss and/orretinal ischemia or eye fibrosis.

In some aspects, the invention provides a method to improve the recoveryfrom a surgery or procedure and/or to treat post-surgical contracture,the method comprising administering a gap junction channel modulator,such as peptide 5, and/or an analogue thereof, compounds of formula I,for example tonabersat, and/or an analogue of any of the foregoingcompounds, to a subject. In a related embodiment, the invention providesthe use of a gap junction channel modulator, such as peptide 5, and/oran analogue thereof, compounds of formula I, for example tonabersat,and/or an analogue of any of the foregoing compounds, in the manufactureof a medicament to improve the recovery from a surgery or procedureand/or to treat post-surgical contracture. In a related embodiment, theinvention provides a gap junction channel modulator, such as peptide 5,and/or an analogue thereof, compounds of formula I, for exampletonabersat, and/or an analogue of any of the foregoing compounds, forexample, to improve the recovery from a surgery or procedure and/or totreat post-surgical contracture.

In some aspects, the invention provides a method to improve the recoveryfrom a surgery or procedure and/or to treat post-surgical contracture,the method comprising administering a pannexin modulator, e.g.,compounds of formula VI, for example probenecid, and/or one or moreanalogue or prodrug of any of the foregoing compounds thereof and/or asynthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1, or ananalogue of either thereof, to a subject. In a related embodiment, theinvention provides the use of a pannexin modulator, e.g., compounds offormula VI, for example probenecid, and/or one or more analogue orprodrug of any of the foregoing compounds thereof and/or a syntheticmimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1, or an analogue ofeither thereof, in the manufacture of a medicament to improve therecovery from a surgery or procedure and/or to treat post-surgicalcontracture. In a related embodiment, the invention provides a pannexinmodulator, e.g., compounds of formula VI, for example probenecid, and/orone or more analogue or prodrug of any of the foregoing compoundsthereof and/or a synthetic mimetic peptide blocker of pannexin 1, e.g.,¹⁰Panx1, or an analogue of either, for example, to improve the recoveryfrom a surgery or procedure and/or to treat post-surgical contracture.In one embodiment, the surgery or procedure is an orthopedic surgery orprocedure. In one embodiment, the post-surgical contracture ispost-orthopedic surgical joint contracture.

In another aspect, the invention provides a method to treat or decreaseadhesion formation in a subject, the method comprising administering agap junction channel modulator, such as peptide 5, and/or an analoguethereof, compounds of formula I, for example tonabersat, and/or ananalogue of any of the foregoing compounds, to a subject. In a relatedembodiment, the invention provides the use of a gap junction channelmodulator, such as peptide 5, and/or an analogue thereof, compounds offormula I, for example tonabersat, and/or an analogue or prodrug of anyof the foregoing compounds, in the manufacture of a medicament to treator decrease adhesion formation in a subject. In a related embodiment,the invention provides a gap junction channel modulator, such as peptide5, and/or an analogue thereof, compounds of formula I, for exampletonabersat, and/or an analogue of any of the foregoing compounds, forexample, to treat or decrease adhesion formation in a subject.

In another aspect, the invention provides a method to treat or decreaseadhesion formation in a subject, the method comprising administering apannexin modulator, e.g., compounds of formula VI, for exampleprobenecid, and/or one or more analogues or prodrugs of any of theforegoing compounds thereof and/or a synthetic mimetic peptide blockerof pannexin 1, e.g., ¹⁰Panx1, or an analogue of either thereof, to asubject. In a related embodiment, the invention provides the use of apannexin modulator, e.g., compounds of formula VI, for exampleprobenecid, and/or one or more analogues or prodrugs of any of theforegoing compounds thereof and/or a synthetic mimetic peptide blockerof pannexin 1, e.g., ¹⁰Panx1, or an analogue of either thereof, in themanufacture of a medicament to treat or decrease adhesion formation in asubject. In a related embodiment, the invention provides a pannexinmodulator, e.g., compounds of formula VI, for example probenecid, and/orone or more analogue or prodrug of any of the foregoing compoundsthereof and/or a synthetic mimetic peptide blocker of pannexin 1, e.g.,¹⁰Panx1, or an analogue of either thereof, for example, to treat ordecrease adhesion formation in a subject.

In another aspect, this invention relates to use of compounds of formulaI, for example tonabersat, and/or an analogue of any of the foregoingcompounds, for example, to directly and immediately block Cx43hemichannels and to cause a concentration and time-dependent reductionin GJ coupling.

In still other aspects, various disorders can be treated by compositionsand methods of the invention, including methods of treatment with a gapjunction channel (e.g., hemichannel) modulator alone or together with apannexin channel modulator. These disorders include but are not limitedto neurodedenerative diseases (e.g. Alzheimer's disease, AIDS-relateddementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, spinal muscular atrophy and cerebellar degeneration);ischemic injury; fibrosis of the lung, kidney or liver; vascular disease(e.g. restenosis, artherosclerosis, atherosclerotic plaques coronaryartery disease, or hypertension), respiratory disease (e.g. asthma,chronic bronchitis, bronchieactasis or cystic fibrosis), undesired oraberrant hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis,psoriatic plaques, sarcoidosis, CNS trauma, including spinal cord injuryand optic nerve injury, diabetic and other proliferative retinopathiesincluding retinopathy of prematurity, retrolental fibroplasia, glaucomaincluding open-angle glaucoma, angle-closure glaucoma, and normotensiveglaucoma, as well as variants of open-angle and angle-closure glaucomasuch as secondary glaucoma, pigmentary glaucoma, pseudoexfoliativeglaucoma, traumatic glaucoma, neovascular glaucoma and irido cornealendothelial syndrome (ICE) and neovascular glaucoma, age-related maculardegeneration, diabetic macular edema, corneal neovascularization,corneal graft neovascularization, corneal graft rejection,retinal/choroidal neovascularization, neovascularization of the angle(rubeosis), ocular neovascular disease, vascular restenosis, uveitis,dry eye disease, ocular and corneal persistent epithelial defects,arteriovenous malformations (AVM), thyroid hyperplasias (includingGrave's disease), corneal and other tissue transplantation, chronicinflammation, lung inflammation, ocular inflammation, acute lunginjury/ARDS, sepsis, systemic capillary leak syndrome, multiple organdysfunction syndrome (MODS), systemic inflammatory response syndrome(SIRS), acute respiratory distress syndrome, post burn vascularpermeability syndromes, conditions marked by ascites, pleural effusionsand pericardial effusions, permeability changes after cardiopulmonarybypass (particularly in infants and young children), reperfusion injury,snakebite, primary pulmonary hypertension, malignant pulmonaryeffusions, cerebral edema (e.g., associated with acute stroke/closedhead injury/trauma), synovial inflammation, osteoarthritis (OA), 3rdspacing of fluid diseases (pancreatitis, compartment syndrome, burns,bowel disease), chronic inflammation such as inflammatory bowel disease(IBD) (including Crohn's disease and ulcerative colitis), renalallograft rejection, inflammatory bowel disease, nephrotic syndrome,hemophilic joints, hypertrophic scars, Osler-Weber syndrome, pyogenicgranuloma retrolental fibroplasias, scleroderma, trachoma, vascularadhesions, synovitis, dermatitis, preeclampsia, ascites, pericardialeffusion (such as that associated with pericarditis), pleural effusion,post surgical tissue edema, acute orthopedic injuries, anaphylaxis,cystic fibrosis, and chronic dermal wounds, ulcers or ulcerous lesions,including diabetic foot ulcers, venous leg ulcers, and pressure ulcers,as well as infectious diseases including malaria, dengue fever, andpneumonia.

Certain preferred treatments for these diseases, disorders andconditions comprise administering a gap junction channel modulator, forexample, compounds of formula I (for example tonabersat), a connexinpeptidomimetic compound, and/or one or more analogue or prodrug of anyof the foregoing compounds thereof, and/or administering a pannexinmodulator, such as a compound of formula VI (for example probenecid)and/or one or more analogue or prodrug of any of the foregoing compoundsthereof, and/or a pannexin peptidomimetic compound and/or one or moreanalogue or prodrug of any of the foregoing compounds thereof.Particularly preferred compounds are the compounds of formula I, forexample tonabersat, and the compounds of formula VI, for exampleprobenecid.

In some aspects the gap junction channel modulator or connexin modulatoris a modulator of a gap junction and/or connexin present in bloodvessels, for example, a connexin43 gap junction channel modulator orconnexin43 modulator, a connexin40 gap junction channel modulator orconnexin40 modulator or a connexin45 gap junction channel modulator orconneixn45modulator. Preferably, the connexin modulator is a connexin 43modulator. Connexin modulators and connexin43 modulators include meansfor down-regulating the connexin transcription or translation of aconnexin, such as antisense molecules, for example. They also includeZO-1 binding peptides in the case of connexin43. Preferably, the gapjunction channel modulator is a connexin43 gap junction channelmodulator. A preferred connexin43 gap junction channel modulator istonabersat, or another compound of Formula I. The pharmaceuticalcompositions of this invention for any of the uses featured herein mayalso comprise a gap junction channel modulator, for example, apolynucleotide, which may inhibit or block, for example, connexin26(Cx26), connexin30 (Cx30), connexin31.1 (Cx31.1), connexin36 (Cx36),connexin37 (Cx37), connexin40 (Cx40), connexin45 (Cx45), connexin 50(Cx50), or connexin 57 (Cx57), or any other connexin, or connexin gapjunction or connexin hemichannel, in the eye or blood vessels. Inanother embodiment, pharmaceutical compositions of this invention forany of the uses featured herein may also be at least one pannexinmodulator, and may further comprise any of the gap junction channelmodulators or connexin modulators described or referenced herein.

In one aspect this invention relates to pharmaceutical compositions,articles of manufacture, and methods for treating ocular and otherdisorders, including for example glaucoma, diabetic retinopathy (DR),diabetic macular edema (DME), age related macular degeneration (AMD),eye fibrosis, and/or neuropathic ocular disorders, by administering atherapeutically effective amount of at least one pannexin modulator tothe eye of said subject. In some aspects the neuropathic ocular disordermay be, for example, glaucoma, loss of RGC and/or glaucomatous ocularneuropathy. In some aspects the glaucoma may result from ocularhypertension. In some aspects, the glaucoma may result from low-tensionor normal-pressure glaucoma. In some aspects, administering atherapeutically effective amount of at least one connexin modulator iseffective for treating (e.g., treating, preventing, slowing, reducing,stopping, or ameliorating) glaucoma, loss of retinal ganglion cells,and/or decreasing vitreal glutamate concentrations. The pharmaceuticalcompositions of this invention for any of the uses featured herein mayalso comprise a pannexin modulator, which may inhibit or block, forexample, pannexin channels. In some aspects the pannexin modulator caninclude or exclude a Panx1, Panx2, or Panx3 modulator. In some aspectsthe pannexin modulator can include or exclude a Panx1 modulator.

In another aspect this invention relates to pharmaceutical compositions,articles of manufacture, and methods for treating ocular and otherdisorders, including for example DR, glaucoma, DME, AMD, eye fibrosis,and/or neuropathic ocular disorders, by administering a therapeuticallyeffective amount of at least one pannexin modulator to the eye of saidsubject. In some aspects the neuropathic ocular disorder may be, forexample, loss of retinal ganglion cells (RGC) and/or glaucomatous ocularneuropathy. In some aspects, administering a therapeutically effectiveamount of at least one pannexin modulator is effective for treating(e.g., treating, preventing, slowing, reducing, stopping, orameliorating) loss of retinal ganglion cells are further useful fordecreasing vitreal glutamate concentrations.

In one aspect this invention relates to pharmaceutical compositions,articles of manufacture, and methods for treating ocular hypertension.In some aspects, treating ocular hypertension treats or preventsintraocular pressure-associated neuropathy. In some aspects the methodfor treating ocular hypertension comprises, for example, administering atherapeutically effective amount of at least one hemichannel or connexinmodulator and/or at least one pannexin channel or pannexin modulator tothe eye of said subject. In one aspect, this invention relates, forexample to pharmaceutical compositions and methods for treatingglaucoma. The methods herein provide for treatment of intraocularpressure-associated optic neuropathy such as glaucoma, in an amountsufficient to reduce intraocular pressure. In some aspects, the connexinmodulators and/or the pannexin modulators are useful in treating traumaassociated with elevated intraocular pressure. In some aspects, theconnexin modulator is a connexin43 modulator. In some aspects, thecompositions and methods of this invention are useful in treating ocularhypertension and reducing the intraocular pressure to normal levels,e.g., below 21 mm Hg, for example, to a level between 8 and 21 mm Hg. Insome aspects, the compositions and methods of this invention are usefulin reducing the intraocular pressure to below about 22 mm, 21 mm, 20 mmHg, or lower.

The compositions, articles of manufacture and methods described hereinare useful, in one aspect, to treat glaucoma without toxic side effects.In some aspects, the glaucoma may be open-angle glaucoma orangle-closure glaucoma. In some aspects, administering a therapeuticallyeffective amount of at least one connexin or hemichannel modulator, forexample, a connexin43 modulator or a connexin43 hemichannel modulator,and/or at least one pannexin modulator to the eye tissue in a subject inneed thereof increases flow through the trabecular meshwork.

In one aspect this invention relates to pharmaceutical compositions,articles of manufacture, and methods for treating normal tension ornormatensive glaucoma. In some aspects this method comprises, forexample, administering a therapeutically effective amount of at leastone connexin or hemichannel modulator and/or at least one pannexinmodulator to the eye of said subject. In one aspect, this inventionrelates, for example to pharmaceutical compositions and methods fortreating glaucoma. In some aspects, the connexin modulator is aconnexin43 or connexin43 hemichannel modulator. In some aspects theconnexin modulator is a modulator of, and can include or exclude, forexample, Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx45, Cx50, Cx57 or anyother connexin or hemichannel comprising said connexin in the eye orblood vessels, and/or at least one pannexin or pannexin channelmodulator. In some aspects, the compositions and methods of thisinvention are useful in treating glaucoma even when intraocular pressureis at normal levels, e.g., below 21 mm Hg. In some embodiments themodulator can include or exclude any of the foregoing.

In some aspects, this invention relates to pharmaceutical compositions,articles of manufacture, and methods for treating ocular disorders thatcan include or exclude glaucoma including hypertensive glaucoma andnormatensive glaucoma, clinical geographic atrophy; AMD including dryAMD and exudative AMD, abnormalities or impairment, chronic macularischemia, fibrosis of the eye, idiopathic polypoidal choroidopathy(IPC); diabetic maculopathy, diabetic retinopathy; hypertensiveretinopathy, inflammatory choroidal neovascularization; central serouschorioretinopathy (CSR); macular telangiectasia; pattern dystrophy;subretinal/subPRD neovascularization; serous detachment of theneurosensory retina; RPE detachment; hemorrhages (subretinal pigmentepithelial, subretinal, intraretinal or pre-retinal, includingbreakthrough bleeding into the vitreous); piretinal, intraretinal,subretinal or sub-pigment epithelial scar/glial tissue or fibrin-likedeposits; retinal fibrosis, retinal angiomatous proliferations andretinochoroidal anatastamosis; choroidal neovascularization (CNV);cystic maculopathy; retinal thickening; non-exudative AMD; and retinalscarring, uveitis, including posterior uveitis, scleritis, episcleritisviral retinitis, including cytomegalovirus (CMV) retinitis, retinopathyof prematurity, retinal hypoxia, diffuse choroidal sclerosis, sclerosisof the choriocapillaris, dry eye, diabetic macular edema (DME),neuropathic ocular disorders, trauma induced lowering of intraocularpressure, epithelial basement membrane dystrophy, and/or other oculardisorders. The compositions and articles of manufacture useful in any ofthe methods of treating described herein comprise a therapeuticallyeffective amount of at least one connexin or hemichannel modulator, forexample, a connexin modulator, or at least one pannexin or pannexinchannel modulator. In some aspects the connexin modulator is a modulatorof, for example, a hemichannel comprising connexin 43 (Cx43), Cx26,Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx45, Cx50, Cx57 or, such connexin, orany other connexin or connexin hemichannel in the eye or blood vessels.In an embodiment for treating diabetic retinopathy, the composition maycomprise, for example, a pannexin or pannexin channel modulator, and/ora small molecule connexin or hemichannel modulator, for example, a smallmolecule inhibitor of connexin43 hemichannels or connexin43 itself. Insome aspects, the connexin modulator for treating diabetic retinopathy,or other diseases, disorders or conditions herein, may be any connexin43modulator of this disclosure, including or excluding polynucleotideshaving SEQ ID NOS.1-3 and/or modified versions thereof. In someembodiments the modulator can include or exclude any of the foregoing.

The inventions described and claimed herein may also be used in thetreatment of cataracts. The central retinal artery provides blood to theeye, with branches to the choroid perfusing the back of the eye andsuperficial and deep vascular plexuses feeding the anterior retina. Inaddition, long branches perfuse the iris and ciliary body to providenutrition and oxygen to the front of the eye, including (via the ciliarybody) the lens. The inventors have surprisingly determined that diseasesaffecting the vascular bed, including diabetic retinopathy and agerelated macular degeneration, will also impact on flow to the anteriorsegment and therefore contribute to cataract formation, and note thatcataracts are one of the most common causes of visual impairment inpeople with diabetes mellitus and that severe cataracts in the eye canalso be associated with a higher prevalence of late AMD.

In some aspects, this invention relates to pharmaceutical compositions,articles of manufacture, and methods for treating ocular disorders thatcan include or exclude macular holes, macular degeneration, retinaltears, DME, diabetic retinopathy (DR), vitreoretinopathy, refractivedisorders, dry eye, viral conjunctivitis, ulcerative conjunctivitis andscar formation in wound healing, corneal epithelial wounds, Sjogren'ssyndrome, cataracts, sequelae of radial keratotomy, increased thicknessof cornea tissue. In this embodiment, the composition may comprise, forexample, a pannexin modulator, or a connexin modulator, for example, asmall molecule inhibitor of connexin 43 or a peptide inhibitor. In someaspects the connexin modulator is a modulator of, for example Cx43, amodulator of Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx 45, Cx50, and Cx57or any other connexin in the eye or blood vessels. In some aspects ofthis embodiment, the compositions may comprise, for example, anyconnexin 43 modulator of this disclosure, including or excludingpolynucleotides having SEQ ID NOS.1-3. In some embodiments the modulatorcan include or exclude any of the foregoing.

In some aspects, this invention relates to pharmaceutical compositions,articles of manufacture, and methods for treating cataracts. In thisembodiment, the composition may comprise, for example, a pannexin orpannexin channel modulator, a gap junction modulator, a gap junction orhemichannel phosphorylation agent, or a connexin antagonist, forexample, polynucleotides, peptides or peptidomimetics or small moleculeantagonists of connexins. In some aspects the connexin modulator is amodulator of Cx43, Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx45, Cx50,Cx57 or any other connexin in the eye or blood vessels. In some aspectsof this embodiment, the compositions may comprise, for example, any Cx43modulator of this disclosure, excluding tonabersat. In some embodimentsthe modulator can include or exclude any of the foregoing.

In some aspects, this invention relates to pharmaceutical compositions,articles of manufacture, and methods for treating ocular disorders thatcan include or exclude retinal vein or artery occlusion, glaucoma,retinal stroke, trauma resulting in raised intraocular pressure,diabetic retinopathy, cystoid macular edema. In this embodiment, thecomposition may comprise, for example, a pannexin or pannexin channelmodulator, a gap junction modulator, a gap junction or hemichannelphosphorylation agent, a ZO-1 binding site peptidomimetic in the case ofCx43, or another connexin antagonist, for example, polynucleotides,peptides or peptidomimetics or small molecule antagonists of connexins.In some aspects the connexin modulator is a modulator of Cx43, Cx26,Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx45, Cx50, Cx57 or any other connexinin the eye or blood vessels. In some aspects of this embodiment, thecompositions may comprise, for example, any Cx43 modulator of thisdisclosure, excluding tonabersat. In some embodiments the modulator caninclude or exclude any of the foregoing.

In some aspects, this invention relates to pharmaceutical compositions,articles of manufacture, and methods for treating Alzheimer's Disease,Parkinson's Disease, Huntington's Disease and Amyotrophic LateralSclerosis, migraine, or aura with or without migraine,sciatica/radiculopathy. In this embodiment, the composition maycomprise, for example, alone or in combination, a pannexin modulator, agap junction or hemichannel phosphorylation agent, or a connexinantagonist, for example. In some aspects the connexin modulator is amodulator of, for example, Cx43, Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40,Cx45, Cx50, Cx57 or any other connexin in the eye or blood vessels. Insome aspects of this embodiment, the compositions may comprise, forexample, any connexin 43 modulator of this disclosure, includingcompounds of formula I, for example tonabersat, and analogs of any ofthe foregoing compounds, as well as, for example, the tonabersatpro-drugs and pro-drugs of the compounds of formula I of this disclosurethat target the eye. In some embodiments the modulator can include orexclude any of the foregoing.

In one aspect this invention relates to pharmaceutical compositions,articles of manufacture, and methods for treating ocular hypoxia, ocularvessel damage and/or blood vessel leakage. In some aspects treating orpreventing ocular hypoxia comprises treating or preventing vesselleakage, vessel breakdown and/or any other condition that results inlower than normal oxygenated blood flow to the choroid or other bloodvessels in the eye. In some aspects treating or preventing ocularhypoxia comprises treating conditions that result in compression ofblood vessels or otherwise impinges on blood flow to the eye. In someaspects the method for treating ocular disorders such as ocular hypoxia,glaucoma, AMD, DME, fibrosis of the eye, and/or retinal perfusiondisorders comprises, for example, administering a therapeuticallyeffective amount of at least one gap junction modulator or connexinmodulator or at least one pannexin or pannexin channel modulator to theeye of said subject. In one aspect, this invention relates, for exampleto pharmaceutical compositions and methods for treating glaucoma or fortreating AMD.

In some aspects, the compositions of this invention are useful asadjuvants to improve trabeculectomy success rates.

The front of the eye is filled with aqueous humor, a clear fluid thatprovides nourishment to the structures in the anterior portion of theeye. This fluid is produced constantly by the ciliary body, whichsurrounds the lens of the eye. Aqueous humor flows through the pupil andout of the eye through the trabecular meshwork channels located at thejunction where the cornea attaches to the iris, which is referred to asthe drainage angle of the eye. In some aspects of this invention, one ormore gap junction or connexin modulators or pannexin or pannexin channelmodulators is administered to the trabecular meshwork or ciliary body.

Also featured in one aspect of this invention are compositions, articlesof manufacture, and methods for treating other ocular conditions, forexample, retinal ischemic diseases or ocular ischemic diseases in asubject, comprising administering a therapeutically effective amount ofa gap junction modulator, a connexin modulator, or a pannexin orpannexin channel modulator, including, for example, amounts effective toreduce inflammation in the inner retina. In some aspects, the retinalischemic disease is retinal artery occlusion, or central retinal veinocclusion. In some aspects, the optic ischemic disease is, for example,anterior ischemic optic neuropathy. In some aspects, the connexinmodulator is a connexin 43 modulator.

This invention also features compositions, articles of manufacture, andmethods for reducing impairment of choridal perfusion and/or choroidalinflammation, or choroidal overperfusion in a subject, comprisingadministering an amount of a gap junction modulator, a connexinmodulator or a pannexin or pannexin channel modulator to the choroid ofthe subject, effective to reduce impairment of choridal perfusion and/orchoroidal inflammation, or choroidal overperfusion in the inner retina.In some aspects of this invention, administering a therapeuticallyeffective amount of a gap junction modulator, a connexin modulator or apannexin or pannexin channel modulator to the choroid of the subjecteffective to reduce impairment of choridal perfusion and/or choroidalinflammation, or choroidal overperfusion, also reduces choriocapillarisendothelial cell loss and/or choriocapillaris dropout, thereby treatingor preventing ocular disorders. In some aspects, reducing impairment ofchoridal perfusion and/or choroidal inflammation, or choroidaloverperfusion also reduces retinal pigmental epithelium degenerationand/or Drusen development, or otherwise ameliorates, stops, slows,and/or reverses the progression of macular degeneration or maculardystrophy, which can be dry macular degeneration or wet maculardegeneration. In some aspects, the gap junction modulator, a connexinmodulator or a pannexin or pannexin channel modulator for reducingimpairment of choridal perfusion and/or choroidal inflammation, orchoroidal overperfusion is a Cx43 modulator. The gap junction modulator,a connexin modulator or a pannexin or pannexin channel modulator forreducing impairment of choridal perfusion and/or choroidal inflammation,or choroidal overperfusion may also be administered with an oculartreatment agent.

Impairment of choridal perfusion and/or choroidal inflammation, orchoroidal overperfusion may induce vascular leak in the choroid andresult in endothelial cell loss in the retinal pigment epithelium. Asdescribed herein, it has been surprisingly discovered that impairment ofchoridal perfusion and/or choroidal inflammation, and choriocapillarisdropout result from Cx43 upregulation, and that Cx43 upregulation is acontributing cause of AMD. Abnormal vasculature was observed in thechoroid of AMD organ donor retinae associated with changes in Cx43expression, supporting the role of Cx43 upregulation in AMD. In someembodiments treating or preventing vascular leak and impairment ofchoridal perfusion and/or choroidal inflammation, or choroidaloverperfusion will be useful in treating or preventing retinal pigmentepithelium degeneration or retinal neovascularisation associated withconditions such as non-exudative AMD, or dry AMD, neovascularization AMDor wet AMD, and Drusen development. As surprisingly discovered anddescribed herein, upstream events such as vascular leak and impairmentof choridal perfusion and/or choroidal inflammation are associated withAMD choroidal changes are then subsequently associated with symptomssuch retinal pigment epithelium degeneration or retinalneovascularisation.

Thus, in one aspect, this invention features methods of administering agap junction modulator, a connexin modulator and/or pannexin or pannexinchannel modulator of this invention alone, or together with one or moreocular treatment agents for use in treating AMD, including ocular agentsfor use in treating neovascularization AMD, wet AMD, Drusen development,dry AMD, retinal pigment epithelium degeneration, or geographic atrophy.The gap junction modulator, connexin modulator or a pannexin or pannexinchannel modulator of this invention may be administered separately,simultaneously, or in a combined composition with the ocular treatmentagents.

In some aspects, the connexin or pannexin oligonucleotides andpolynucleotides of this invention are made chemically, synthetically, orotherwise manufactured. In some embodiments the connexin modulator is aconnexin oligonucleotide or polynucleotide. In one embodiment theconnexin modulator is a connexin antisense oligodeoxynucleotide, whetherchemically modified or unmodified, for example, a Cx43 antisenseoligodeoxynucleotide or an antisense oligodeoxynucleotide to Cx26, Cx30,Cx31.1, Cx36, Cx37, Cx40, Cx45, Cx50, Cx57 or any other connexin in theeye or blood vessels. In some aspects modified connexin antisensepolynucleotides or oligonucleotides may comprise mixtures of modifiedand unmodified nucleotides. In some aspects, the connexin antisensecompound used in the methods herein is an antisense oligonucleotidecomprising naturally occurring nucleotides and unmodifiedinternucleoside linkages. In some embodiments the connexinoligonucleotide or polynucleotide and/or connexin antisenseoligodeoxynucleotide may be, for example, a Cx43 antisenseoligonucleotide, polynucleotide, and/or Cx43 antisenseoligodeoxynucleotide. In some embodiments the modulator can include orexclude any of the foregoing.

Featured in this invention are connexin antisense oligonucleotides orpolynucleotides comprising at least one unmodified nucleotide. In oneaspect, the connexin antisense oligonucleotides or polynucleotides maycomprise at least one modified nucleotide, and/or have at least onemodified internucleoside linkage, and/or at least one modified sugarmoiety. The modified internucleoside linkage may be, for example, aphosphorothioate linkage. In some aspects, for example, the connexinpolynucleotide may comprise at least one nucleotide comprising aconformationally strained nucleotide, for example, a locked nucleic acid(LNA) or a bridged nucleic acid (BNA). The locked nucleotide may beselected, from one of the following types, for example: 2′-O—CH₂-4′(oxy-LNA), 2′-CH₂—CH₂-4′ (methylene-LNA), 2′-NH—CH₂-4′ (amino-LNA),2′-N(CH₃)—CH₂-4′ (methylamino-LNA), 2′-S—CH₂-4′ (thio-LNA), and2′-Se—CH₂-4′ (seleno-LNA). In some aspects the modified nucleotide maybe a locked nucleic acid or an unlocked nucleic acid. In some aspectsthe modified and unmodified connexin antisense oligonucleotides orpolynucleotides are modified and unmodified Cx43 antisenseoligonucleotides or polynucleotides. In some aspects the modified andunmodified connexin antisense oligonucleotides or polynucleotides aremodified and unmodified Cx26 antisense oligonucleotides orpolynucleotides, Cx31.1 antisense oligonucleotides or polynucleotides,Cx30 antisense oligonucleotides or polynucleotides, Cx45 antisenseoligonucleotides or polynucleotides, Cx36 antisense oligonucleotides orpolynucleotides, Cx37 antisense oligonucleotides or polynucleotides,Cx40 antisense oligonucleotides or polynucleotides, Cx50 antisenseoligonucleotides or polynucleotides, or Cx57 antisense oligonucleotidesor polynucleotides. In some aspects, the connexin antisenseoligonucleotides or polynucleotides comprise chiral phosphorous moietiessuch as those described in WO2013012758.

Also featured herein are exemplary modified or unmodified connexin 43antisense compounds comprising a nucleotide sequence or modified from anucleotide sequence selected from SEQ ID NO:1-16. The polynucleotides ofthis invention include polynucleotides having a length of less than 80nucleotides, e.g., from 12-18 to about 50-80 nucleotides, preferablyabout 30 nucleotides or less, e.g., from 12 to about 30 nucleotides, andmore preferably from about 15 to about 30 nucleotides. In one example,the polynucleotide has 30 nucleotides. The methods of this inventionfeatures, in some aspects, the use of connexin 43 antisense compounds upto 40 nucleotides in length, for example, 15 to 40 nucleotides inlength, comprising a nucleotide sequence selected from SEQ ID NOs:1-17,from SEQ ID NOs:4-17, or comprising from about 8 to 40 nucleotides ofSEQ ID NO:17. In some embodiments the Cx43 antisense compounds may bemodified by substituting one or more uridine nucleotides residues forone or more thymine nucleotides in SEQ ID NOs:4-17, or in SEQ ID Nos1-3.

Also featured herein are modified or unmodified Cx45 antisensepolynucleotides comprising from 8 to about 80 nucleotides of SEQ. ID.NO: 217 and modified and unmodified pannexin antisense polynucleotidescomprising from 8 to about 80 nucleotides of SEQ. ID. NO: 279. Thepolynucleotides of this invention include synthesized polynucleotideshaving a length of less than 80 nucleotides, e.g., from 12-18 to about50-80 nucleotides, preferably about 30 nucleotides or less, e.g., from12 to about 30 nucleotides, and more preferably from about 15 to about30 nucleotides. In one example, the polynucleotide has 30 nucleotides.The methods of this invention features, in some aspects, the use ofconnexin 45 antisense compounds up to 40 nucleotides in length, forexample, 15 to 40 nucleotides in length, for example, comprising fromabout 8 to about 40 or from about 15 to about 40 nucleotides of SEQ IDNO:217. The methods of this invention features, in some aspects, the useof pannexin antisense compounds up to 40 nucleotides in length, forexample, 15 to 40 nucleotides in length, for example, comprising fromabout 8 to about 40 or from about 15 to about 40 nucleotides of SEQ IDNO:117-121. In some embodiments the connexin 45 or pannexin antisensecompounds may be modified by substituting one or more uridinenucleotides residues for one or more thymine nucleotides in SEQ IDNO:217, or SEQ ID NO:117-121.

Also featured for use in any of the methods featured herein are modifiedor unmodified Cx26 polynucleotides, Cx31.1 polynucleotides, Cx36polynucleotides, Cx37 polynucleotides, Cx40 polynucleotides, Cx50polynucleotides, or Cx57 polynucleotides or modified or unmodifiedanti-sense polynucleotides to any other connexin in the eye or bloodvessels. The pharmaceutical compositions of this invention for any ofthe uses featured herein may also comprise a pannexin antisensepolynucleotide modulator, which may inhibit pannexin channels, forexample, Panx1 or Panx 2, or Panx3. In some embodiments the modulatorcan include or exclude any of the foregoing.

In some aspects of this invention, the connexin 43 antisenseoligonucleotide or polynucleotide has at least about 80%, 85%, 90%, 95%,97%, 98%, or 99% homology to a polynucleotide having a sequence selectedfrom SEQ ID NOs: 1 to 17. Connexin or pannexin modulators that areoligonucleotides or polynucleotides may have at least about 80%, 85%,90%, 95%, 97%, 98%, or 99% homology to an 8 to 80 nucleotide portion oftheir respective sequences. For example, connexin 45 modulators that areoligonucleotides or polynucleotides may have at least about 80%, 85%,90%, 95%, 97%, 98%, or 99% homology to an 8 to 80 nucleotide portion ofSEQ ID NO:217, while pannexin modulators that are oligonucleotides orpolynucleotides may have at least about 80%, 85%, 90%, 95%, 97%, 98%, or99% homology to an 8 to 80 nucleotide portion of SEQ ID NO: 117 (Panx1polynucleotide), (Panx1 polynucleotide RefSeq ID NM_015368.3), SEQ IDNO:118 (Panx2 polynucleotide), (Panx2 polynucleotide RefSeq IDNM_052839.3 for variant 1), SEQ ID NO:119 (RefSeq ID NM_001160300.1 forPanx2 polynucleotide variant 2), SEQ ID NO: 120 (RefSeq ID NR_027691.1for Panx2 polynucleotide variant 3), or SEQ ID NO: 121 (Panx3polynucleotide) (Panx3 polynucleotide RefSeq ID NM_052959.2), orvariants thereof. In some aspects, the pannexin modulators can includeor exclude pannexin peptide sequences. The pannexin peptide sequencescan comprise 8-40 consecutive amino acids, an extracellular domain, anintracellular domain, a carboxy terminus part, or an amino terminuspart, of the polypeptides SEQ ID NO: 122 (Panx1 peptide), SEQ ID NO: 123(Panx2 peptide), or SEQ ID NO: 124 (Panx3 peptide), or variants thereof.In some embodiments the modulator can include or exclude any of theforegoing.

In other embodiments, the gap junction modulators or connexin modulatorsare connexin peptides or peptidomimetics, sometimes referred toanti-connexin peptides or peptdomimetics, e.g., anti-connexinhemichannel blocking peptides or peptidomimetics, for example, modifiedor unmodified peptides or peptidomimetics comprising connexinextracellular domains, transmembrane regions, and connexincarboxy-terminal peptides). The gap junction modulators or anti-connexinhemichannel blocking peptides or peptidomimetics may be modified orunmodified. The gap junction modulators and anti-connexin hemichannelblocking peptides or peptidomimetics are made chemically, synthetically,or otherwise manufactured. In some embodiments, the gap junctionmodulators or connexin modulators are Cx43 peptides or peptidomimetics.In some aspects, the therapeutically effective modified or unmodifiedpeptide or peptidomimetic comprises a portion of an extracellular ortransmembrane domain of a connexin, such as Cx43 or Cx45. In otherembodiments, the pannexin or pannexin channel modulators are pannexinpeptides or peptidomimetics, sometimes referred to anti-pannexinpeptides or peptidomimetics, for example, modified or unmodifiedpeptides or peptidomimetics.

In some embodiments the modulators of this invention include anti-Cx43peptides or peptidomimetics, for example, any of the peptides describedherein, including peptides comprising a portion of an extracellulardomain of a connexin, and peptides comprising a portion of acarboxy-terminal portion of a connexin useful in the methods of thisinvention, which is therapeutically effective, for example, effectivefor healing any of the neuropathic ocular disorders described herein. Insome aspects, the therapeutically effective modified or unmodifiedpeptide or peptidomimetic comprises a portion of an extracellular ortransmembrane domain of a connexin, such as Cx43.

In some embodiments the modulator may be gap junction closing compoundsand hemichannel closing compounds. In some embodiments, the gap junctionclosing compounds and hemichannel closing compounds are connexin 43 gapjunction closing compounds and connexin 43 hemichannel closingcompounds. Preferred connexin carboxy-terminal polypeptides are connexin43 carboxy-terminal polypeptides.

In other embodiments, the gap junction modulators are gap junctionpeptides or peptidomimetics, sometimes referred to anti-gap junctionpeptides or peptdomimetics, e.g., anti-gap junction or hemichannelblocking peptides or peptidomimetics, for example, modified orunmodified peptides or peptidomimentics comprising extracellulardomains, transmembrane regions, or connexin carboxy-terminal peptides ofproteins comprising gap junctions in the eye, including Cx43, Cx26,Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx45, Cx50, and Cx57. The anti-gapjunction or hemichannel blocking peptides or peptidomimetics may bemodified or unmodified. The anti-gap junction or hemichannel blockingpeptides or peptidomimetics are made chemically, synthetically, orotherwise manufactured. In some embodiments, the gap junction modulatorsare Cx43, Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx45, Cx50, and Cx57peptides or peptidomimetics.

Treatment of a subject for any of the ocular conditions describedherein, e.g., for glaucoma, DME, eye fibrosis, AMD, or other oculardisorders as referenced herein with one or more pharmaceuticalcompositions of the invention, e.g., an anti-connexin ODN and a gapjunction modulator such as a connexin hemichannel blocking agent, e.g.,a peptide or peptidomimetic, or a first anti-connexin agent and a secondanti-connexin agent, may comprise their simultaneous, separate,sequential or sustained administration.

In some aspects of this invention, the modulator is a gap junctionclosing or blocking compound or hemichannel closing or blockingcompound, such as tonabersat. In some embodiments, the gap junctionmodulator can be a small molecule, which may also be referred to hereinas an anti-connexin or connexin modulator. In some aspects, theanti-connexin modulator drug can have the structure in Formula I:

wherein Y is C—R₁;

R₁ is acetyl;

R₂ is hydrogen, C₃₋₈ cycloalkyl, C₁₋₆ alkyl optionally interrupted byoxygen or substituted by hydroxy, C₁₋₆ alkoxy or substitutedaminocarbonyl, C₁₋₆ alkylcarbonyl, C_(1_6) alkoxycarbonyl, C₁₋₆alkylcarbonyloxy, C₁₋₆ alkoxy, nitro, cyano, halo, trifluoromethyl, orCF3S; or a group CF₃-A-, where A is —CF₂—,

—CO—, —CH₂—, CH(OH), SO₂, SO, CH₂—O, or CONH; or a group CF₂H-A′- whereA′ is oxygen, sulphur, SO, SO₂, CF₂ or CFH; trifluoromethoxy, C₁₋₆alkylsulphinyl, perfluoro C₂₋₆ alkylsulphonyl, C₁₋₆ alkylsulphonyl, C₁₋₆alkoxysulphinyl, C₁₋₆ alkoxysulphonyl, aryl, heteroaryl, arylcarbonyl,heteroarylcarbonyl, phosphono, arylcarbonyloxy, heteroarylcarbonyloxy,arylsulphinyl, heteroarylsulphinyl, arylsulphonyl, orheteroarylsulphonyl in which any aromatic moiety is optionallysubstituted, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkoxycarbonylamino, C₁₋₆alkyl-thiocarbonyl, C₁₋₆ alkoxy-thiocarbonyl, C₁₋₆alkyl-thiocarbonyloxy, 1-mercapto C₂₋₇ alkyl, formyl, or aminosulphinyl,aminosulphonyl or aminocarbonyl, in which any amino moiety is optionallysubstituted by one or two C₁₋₆ alkyl groups, or C₁₋₆alkylsulphinylamino, C₁₋₆ alkylsulphonylamino, C₁₋₆ alkoxysulphinylaminoor C₁₋₆ alkoxysulphonylamino, or ethylenyl terminally substituted byC₁₋₆ alkylcarbonyl, nitro or cyano, or —C(C₁₋₆ alkyl)NOH or —C(C₁₋₆alkyl)NNH₂; or amino optionally substituted by one or two C₁₋₆ alkyl orby C₂₋₇ alkanoyl; one of R₃ and R₄ is hydrogen or C₁₋₄ alkyl and theother is C₁₋₄ alkyl, CF₃ or CH₂X^(a) is fluoro, chloro, bromo, iodo,C₁₋₄ alkoxy, hydroxy, C₁₋₄ alkylcarbonyloxy, —S—C₁₋₄ alkyl, nitro, aminooptionally substituted by one or two C₁₋₄ alkyl groups, cyano or C₁₋₄alkoxycarbonyl; or R₃ and R₄ together are C₂₋₅ polymethylene optionallysubstituted by C₁₋₄ alkyl;

R₅ is C₁₋₆ alkylcarbonyloxy, benzoyloxy, ONO₂, benzyloxy, phenyloxy orC₁₋₆ alkoxy and R₆ and R₉ are hydrogen or R₅ is hydroxy and R₆ ishydrogen or C₁₋₂ alkyl and R₉ is hydrogen;

R₇ is heteroaryl or phenyl, both of which are optionally substituted oneor more times independently with a group or atom selected from chloro,fluoro, bromo, iodo, nitro, amino optionally substituted once or twiceby C₁₋₄ alkyl, cyano, azido, C₁₋₄ alkoxy, trifluoromethoxy andtrifluoromethyl;

R₈ is hydrogen, C₁₋₆ alkyl, OR₁₁ or NHCOR₁₀ wherein R₁₁ is hydrogen,C₁₋₆ alkyl, formyl, C₁₋₆ alkanoyl, aroyl or aryl-C₁₋₆ alkyl and R₁₀ ishydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, mono or di C.sub.1-6 alkyl amino,amino, amino-C.sub.1-6 alkyl, hydroxy-C₁₋₆ alkyl, halo-C₁₋₆ alkyl, C₁₋₆acyloxy-C₁₋₆ alkyl, C₁₋₆ alkoxycarbonyl-C_(1_6)-alkyl, aryl orheteroaryl; the R₈—N—CO—R₇ group being cis to the R₅ group; and X isoxygen or NR₁₂ where R₁₂ is hydrogen or C₁₋₆ alkyl.

For any of the Markush groups set forth above, in some embodiments, eachgroup can include or exclude any of the species listed for that group.

In some embodiments, the small molecule connexin modulator can beTonabersat, carabersat, or SB-204269. SB-204269 is also known as(trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3R-ol). Carabersat is also knownasN-[(3R,4S)-6-Acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-chromen-4-yl]-4-fluorobenzamide.Tonabersat is also known asN-(6-Acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-chromen-4-yl)-3-chloro-4-fluorobenzamide.

For any of the Markush groups set forth above, that group can include orexclude any of the species listed for that group.

In some embodiments, the connexin modulator may be a pro-drug of any thecompounds for use in this invention. In one aspect the connexinmodulator pro-drug of this invention may be a compound of Formula II:

wherein

Q is O or an oxime,

R₂ is H,

A is a direct bond, —C(O)O*—, —C(R₃)(R₄)O*—, —C(O)O—C(R₃)(R₄)O*—, or—C(R₃)(R₄)OC(O)O*— wherein the atom marked * is directly connected toR₁, R₃ and R₄ are selected independently from H, fluoro, C₁₋₄ alkyl, orC₁₋₄ fluoroalkyl, or R₃ and R₄ together with the atom to which they areattached form a cyclopropyl group,

R₁ is selected from groups [1], [2], [2A], [3], [4], [5] or [6] whereinthe atom marked ** is directly connected to A:

R₅ and R₆ are each independently selected from H, C₁₋₄ alkyl, C₁₋₄fluoroalkyl, or benzyl;

R₇ is independently selected from H, C₁₋₄ alkyl, or C₁₋₄ fluoroalkyl;

R₈ is selected from:

(i) H, C₁₋₄ alkyl, or C₁₋₄ fluoroalkyl, or

(ii) the side chain of a natural alpha-amino acid, or

(iii) biotin or chemically linked to biotin;

R₉ is selected from H, —N(R₁₁)(R₁₂), or —N⁺(R₁₁)(R₁₂)(R₁₃)X⁻, or—N(Ru)C(O)R₁₄

wherein R₁₁, R₁₂, and R₁₃ are independently selected from H, C₁₋₄ alkyl,or C₁₋₄ fluoroalkyl,

R₁₄ is H, C₁₋₄ alkyl, or C₁₋₄ fluoroalkyl,

R₁₅ is selected from C₁₋₄ alkyl or C₁₋₄ fluoroalkyl, and

X⁻ is a pharmaceutically acceptable anion.

In some aspects,

R₂ is B—R₂₁ wherein,

B is a direct bond, —C(O)O*—, —C(R₂₃)(R₂₄)O*, C(O)OC(R₂₃)(R₂₄)*, or

C(R₂₃)(R₂₄)OC(O)O* wherein the atom marked * is directly connected toR₂₁,

R₂₃ and R₂₄ are selected independently from H, fluoro, C₁₋₄ alkyl, orC₁₋₄ fluoroalkyl,

R₂₁ is selected from groups [21], [22], [22A], [23], [24], [25] and [26]wherein the atom marked ** is directly connected to B:

wherein R₅, R₆, R₇, R₈, R₉, and R₁₅ are as defined herein.

For any of the Markush groups set forth above, that group can include orexclude any of the species listed for that group.

In some aspects, the peptide as described herein can be a connexinmodulator, calmodulin modulator, or pannexin modulator.

In some aspects,

Q is an oxime of formula ═NHOR₄₃, wherein R₄₃ is

(i) selected from H, C₁₋₄ fluoroalkyl or optionally substituted C₁₋₄alkyl, or

(ii) -A₃₀₀-R₃₀₀ wherein

A₃₀₀ is a direct bond, —C(O)O*—, —C(R₃)(R₄)O*—, —C(O)O—C(R₃)(R₄)O*—, or—C(R₃)(R₄)OC(O)O*— wherein the atom marked * is directly connected toR₃₀₀,

R₃ and R₄ are selected independently from H, fluoro, C₁₋₄ alkyl, or C₁₋₄fluoroalkyl, or

R₃ and R₄ together with the atom to which they are attached form acyclopropyl group,

R₃₀₀ is selected from groups [1], [2], [2A], [3], [4], [5] or [6]wherein the atom marked ** is directly connected to A₃₀₀:

R₅ and R₆ are each independently selected from H, C₁₋₄ alkyl, C₁₋₄fluoroalkyl, or benzyl;

R₇ is independently selected from H, C₁₋₄ alkyl, or C₁₋₄ fluoroalkyl;

R₈ is selected from:

(iii) H, C₁₋₄ alkyl, or C₁₋₄ fluoroalkyl, or

(iv) the side chain of a natural alpha-amino acid, or

(v) biotin or chemically linked to biotin;

R₉ is selected from H, —N(R₁₁)(R₁₂), or —N⁺(R₁₁)(R₁₂)(R₁₃)X⁻, or—N(R₁₁)C(O)R₁₄

wherein R₁₁, R₁₂, and R₁₃ are independently selected from H, C₁₋₄ alkyl,or C₁₋₄ fluoroalkyl,

R₁₄ is H, C₁₋₄ alkyl, or C₁₋₄ fluoroalkyl,

R₁₅ is selected from C₁₋₄ alkyl or C₁₋₄ fluoroalkyl, and

X⁻ is a pharmaceutically acceptable anion.

In an embodiment R₄₃ is C₁₋₄ alkyl optionally substituted with aphosphate group (P(O)OR₆₁R₆₂). In an example of such an embodiment OR₄₃is —OCH₂P(O)OR₆₁OR₆₂, wherein R₆₁ and R₆₂ are independently H or C₁₋₄alkyl.

In another embodiment R₄₃ is an amino acid derivative having thestructure C(O)CH(R₁₀₀)NH₂ wherein the group R₁₀₀ is the side chain of anatural or unnatural amino acid or a peptide as described herein.

In an embodiment OR₄₃ is —OC(O)CH(CH(CH₃)₂)NH₂.

For any of the Markush groups set forth above, in some embodiments, eachgroup can include or exclude any of the species listed for that group.

In some aspects, the peptide as described herein can be any of thepeptide or peptidomimetic modulators disclosed herein, for example, apeptide or peptidomimetic connexin modulator or pannexin modulator. Insome aspects the peptide connexin modulator can be any of SEQ ID NOs:140-200, 72-139, 216-224, 244-256 and 132.

The compositions described herein can be used to an ocular conditionincluding or excluding those described herein.

In some aspects, the pro-drug can be those described in WO 2014/006407,also published as US 2015/0322034, and herein incorporated by reference.In some aspects the promoeity of the connexin modulator may comprise achaperone moeity that targets one or more regions or structures of theeye. The promoeity may be any peptidomimetic or peptide antagonist ofthis disclosure. In some embodiments, the promoeity can be a singleamino acid which is optionally protected on its functional groups. Insome embodiments, the promoeity can be a targeting species. In someaspects, the promoeity can be a substrate for an influx or effluxtransporters on the cell membrane. The promoeity can be, for example,chemically-linked biotin. The promoeity can be, for example,chemically-linked D-serine.

In one aspect of the methods of this invention, one or more gap junctionmodulators, one or more pannexin channel modulators, one or morehemichannel modulators, one or more connexin modulators, or one or morepannexin modulators as described herein may be administered incombination with one or a plurality of ocular treatment agents usefulfor treating ocular disorders, including, for example, glaucoma, ocularfibrosis, ocular hypoxia, AMD, DME, ocular hypoxia, and/or neuropathicdisorders of the eye. In some aspects of this invention, the one or moreconnexin modulators or pannexin modulators, administered for thetreatment of ocular disorders, for example, wet or dry AMD, neuropathicocular disorders such as intraocular loss of retinal ganglion cellsglaucomatous ocular neuropathy and/or intraocular pressure-associatedneuropathy, may be administered with an ocular treatment agent. In someembodiments the one or more gap junction modulators, one or morepannexin channel modulators, one or more hemichannel modulators, one ormore connexin modulators or more pannexin modulators may beco-administered in a formulation comprising the one or more gap junctionmodulators, one or more pannexin channel modulators, one or morehemichannel modulators, one or more connexin modulators or more pannexinmodulators and the ocular treatment agent.

Ocular treatment agents for use in combination with one or more gapjunction modulators, one or more pannexin channel modulators, one ormore hemichannel modulators, one or more connexin modulators or one ormore pannexin modulators of this invention include, for example,anti-VEGF modulating agents such as VEGF antagonists, mTOR inhibitors,PDGF modulators such as PDGF antagonists, inhibitors of S1P production,squalamine, PEDF producers, tubulin binding agents, integrin inhibitors,or other therapeutic agents useful, in treating, for example,neovascularation AMD or wet AMD. Preferably, the ocular treatment agentfor use in combination with one or more of the modulators of thisinvention is a PDGF modulator. In some aspects, the modulator caninclude or exclude any of the foregoing.

In some aspects, VEGF modulators can be antagonists that inhibit and/orblock VEGF or that inhibit and/or block upstream agonists of VEGF. Insome aspects the VEGF antagonists include, for example, antagonists thatbind to and inhibit VEGF, compounds that inhibit expression of VEGF,and/or viral vectors comprising VEGF inhibitors or encoding proteins orantisense polynucleotides that block or inhibit VEGF. In some aspects,species that inhibit VEGF and/or upstream agonists of VEGF can beantibodies or antibody fragments, nanobodies, peptide orpeptidomimetics, receptor fragments, recombinant fusion proteins,aptamers, small molecules, or single chain variable fragments (scFv). Insome aspects, VEGF antagonist antibodies can be, for example, Lucentis™(ranibizumab), and/or Avastin™ (bevacizumab).

Increased mTOR activity in cells can lead to secretion of VEGF and PDGF,which promotes angiogenesis by increasing mTOR activity in vascularcells. In some aspects of this invention, the VEGF modulators decreasethe activity of VEGF-R or mTOR, thereby reducing angiogenesis. In someaspects, VEGF antagonists that inhibit and/or block upstream agonists ofVEGF binding partners and inhibit VEGF can be a RTP801 inhibitor orREDD1 blocker. In addition, increased mTOR activity in cells can lead tosecretion of VEGF and PDGF, which promotes angiogenesis by increasingmTOR activity in vascular cells. Accordingly, in some aspects, the VEGFmodulators can be mTOR inhibitors, for example, macrolides or smallmolecules.

Ocular treatment agents for use in combination with one or more of thegap junction modulators, one or more pannexin channel modulators, one ormore hemichannel modulators, one or more connexin modulators or one ormore pannexin modulators of this invention also include, for example,complement modulators, and other therapeutic agents useful, for example,in treating geographic atrophy, dry AMD, non-exudative AMD, and/orDrusen development. The complement modulators can be, for example,compstatin, TP10, Eculizumab, ARC1905, JPE-1375, PMX53, Lampalizumab, orrhCFHp.

In some aspects, ocular treatment agents for use in combination with oneor more of the gap junction modulators, one or more pannexin channelmodulators, one or more hemichannel modulators, one or more connexinmodulators or one or more pannexin modulators of this invention caninclude TNF-alpha inhibitors, C-raf kinase inhibitors, NSAIDs, or nAChRinhibitors.

The anti-connexin modulator used in any of the administration,co-administrations, compositions, kits or methods of treatment of thisinvention may be a Cx43 modulator, a Cx45 modulator, a modulator ofCx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx50, and Cx57 or a modulator ofany connexin present in ocular (or other) blood vessels.

The ocular treatment agent useful in treating an ocular disorder, suchas glaucoma and/or retinal perfusion impairment, wet and/or dry AMD, orany other ocular disorder referenced herein, may be, for example, analpha 2 agonist, such as brimonidine, a carbonic anyhydrase inhibitor, abeta blocker, F2a prostaglandin analogs, anti-apoptosis agents,N-methyl-D-aspartate (NMDA) receptor antagonists, a Rho kinaseinhibitor, or glutamate release inhibitors. The ocular treatment agentmay also be a combination therapy comprising administering two or moreocular treatment agents, for example can include or exclude, a betablocker and a carbonic anhydrase inhibitor, such as timolol & travoprostor timolol & dorzolamide, a combination of an alpha 2 agonist and abeta-blocker such as brimonidine and timolol, or a combination of analpha 2 agonist and a carbonic anyhydrase inhibitor such as brinzolamideand brimonidine. In some aspects, the ocular treatment agents mayinclude or exclude rho kinase inhibitors. In other aspects the oculartreatment agents may be adenosine mimetics. Other ocular treatmentagents may include neurotrophins such as ciliary neurotropic factor ornerve growth factor. In some aspects the ocular treatment agent caninclude or exclude one or more of alibercept, lampalizumab,sonepcizumab, fenretinide, ranibizumab (e.g., Lucentis™), bevacizumab(e.g., Avastin™), protein ciliary neutrophic factor, a vascularendothelial growth factor-modulating compound, and a hypoxia-induciblefactor 1-alpha-modulating compound, and any mixture thereof. In oneaspect, the gap junction modulator, pannexin channel modulator,hemichannel modulator, connexin modulator or pannexin modulatoradministered alone or in combination with one or more ocular treatmentagents may be modified or unmodified. In some aspects of this invention,a modified connexin or pannexin modulator may comprise modified andunmodified moieties, such as modified and unmodified nucleotides ormodified and unmodified amino acids. In some aspects, the connexin orother modulator, may be, for example, a modified oligonucleotide, amodified polynucleotide, or a modified peptide or peptidomimetic, asdescribed herein. In some aspects, the connexin modulator may beco-administered before, with or after, or on the same or paralleladministration schedules as, or in the same formulation as the one ormore ocular treatment agents. In some aspects, the pannexin or othermodulator, may be, for example, a modified oligonucleotide, a modifiedpolynucleotide, or a modified peptide or peptidomimetic, as describedherein. In some aspects, the pannexin or other modulator may beadministered before, with or after, or on the same or paralleladministration schedules as, the one or more ocular neuropathictreatments. In some aspects, the pannexin or other modulator, may be,for example, a modified oligonucleotide, a modified polynucleotide, or amodified peptide or peptidomimetic, as described herein. In someaspects, the pannexin or other modulator may be administered before,with or after, or on the same or parallel administration schedules as,the one or more ocular neuropathic treatments.

In some aspects of the methods of this invention, two or more gapjunction modulators, hemichannel modulators, or such as two or moreconnexin modulators as described herein may be administered, which canbe the same or a different type of modulator, for example, two or morepolynucleotides, two or more peptides or peptidomimetic compounds, ortwo or more compounds. For example, in some aspects of the methods ofthis invention, two or more Cx43 modulators as described herein may beadministered, which can be the same or a different type of modulator,for example, two or more polynucleotides, two or more peptides orpeptidomimetic compounds, or two or more compounds. In some embodiments,for example, one or more Cx43 polynucleotides may be co-administeredwith one or more Cx43 peptides or peptidomimetics and/or one or moreanti-connexin compounds, or any subcombination thereof. In some aspects,the methods of this invention may comprise administering an connexinanti-sense polynucleotide to transiently modulate gap junction channels:by modulating protein expression, and further comprise administering aconnexin peptide or peptidomimetic to directly and more immediatelymodulate gap junction channel function. For example, the methods of thisinvention may comprise administering an Cx43 anti-sense polynucleotideto transiently modulate gap junction channels: by modulating proteinexpression, and further comprise administering a Cx43 peptide orpeptidomimetic to directly modulate gap junction channel function. Themodulators may also be targeted to the same or different proteins, suchas Cx43, Cx45 and/or a pannexin. The connexin modulators and/orblockers, pannexin modulators and/or blockers, gap junction modulators,hemichannel modulators and/or blockers may be modified or unmodified.

In some aspects of the methods of this invention, one or more gapjunction and/or connexin modulators as described herein may beadministered with a pannexin modulator, which can be the same or adifferent type of modulator, for example, two or more polynucleotides,two or more peptides or peptidomimetic compounds, or two or morecompounds. In some aspects the connexin modulator is a Cx43 modulator.

In some aspects of the methods of this invention, two or more pannexinor pannexin channel modulators as described herein may be administered,which can be the same or a different type of modulator, for example, twoor more polynucleotides, two or more peptides or peptidomimeticcompounds, or two or more compounds.

As described further herein, the modified oligonucleotide furthercomprises one or more of the following selected components: a modifiedinternucleoside linkage, for example, a phosphorothioate linkage, and amodified sugar moiety, for example, a conformationally-strained sugar,for example, an LNA or BNA.

In some aspects of this invention the connexin modulators (for example,Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, or Cx57modulators) or pannexin modulators of the invention are combined with apharmaceutically acceptable carrier or diluent to produce apharmaceutical composition. In some aspects, suitable carriers anddiluents include buffered, aqueous solutions, isotonic saline solutions,for example phosphate-buffered saline, isotonic water, and the like.

In another aspect, a P2X7 receptor antagonist is administered alone ortogether with a gap junction modulator, a pannexin channel modulator, ahemichannel modulator, a connexin modulator and/or a pannexin modulatorto treat the diseases, disorders and conditions referenced herein. P2X7antagonists include NF279 suramin analog), calmidazolium (a calmodulinantagonist), KN-62 (a CaM kinanse II antagonist), zinc, calcium,magnesium and copper.

Various preferred embodiments include use of a small molecule thatblocks or ameliorates or otherwise antagonizes or inhibits hemichannelopening, alone or together with a small molecule that blocks orameliorates or otherwise antagonizes or inhibits pannexin channelopening, to treat the diseases, disorders and conditions referencedherein. In various preferred embodiments, the small molecule that blocksor ameliorates or inhibits hemichannel opening is tonabersat or ananalogue thereof, or a prodrug of either. In various embodiments, thesmall molecule that blocks or ameliorates or inhibits pannexin channelopening is probenecid or an analogue thereof, or a prodrug of either. Invarious preferred embodiments, the disease, disorder or condition is anocular neovascular disease, an ocular edema, an ocular microvasculardisorder, and a diabetic ocular disease. In certain embodiments, thedisease, disorder or condition is diabetic retinopathy, ischemicretinopathy, choroidal neovascularization, iris neovascularization,corneal neovascularization, retinal neovascularization, intraocularneovascularization, wet age-related macular degeneration, dryage-related macular degeneration, ocular geographic atrophy, diabeticmacular edema, diabetic retinopathy, diabetic retinal ischemia,proliferative diabetic retinopathy, Coates disease, central retinal veinocclusion (CRVO), branched central retinal vein occlusion (BRVO),retinopathy of prematurity (ROP), subconjunctival hemorrhage, andhypertensive retinopathy, uveitis or cataracts.

In other preferred embodiments, the disease, disorder or condition isglaucoma, including open-angle glaucoma, angle-closure glaucoma, andnormotensive glaucoma, as well as variants of open-angle andangle-closure glaucoma such as secondary glaucoma, pigmentary glaucoma,pseudoexfoliative glaucoma, traumatic glaucoma, neovascular glaucoma andirido corneal endothelial syndrome (ICE) and neovascular glaucoma. Inanother preferred embodiments, the disease, disorder or condition is dryeye disease. In another embodiment, the disease, disorder or conditionis ocular and corneal persistent epithelial defect. In still otherpreferred embodiments, the disease, disorder or condition is a chronicdermal wound, ulcer or ulcerous lesion, including diabetic foot ulcers,venous leg ulcers, and pressure ulcers. In another preferred embodiment,the disease, disorder or condition is a CNS trauma, including spinalcord injury and optic nerve injury. The invention also features a methodfor treating a patient diagnosed with or at risk for developing aneovascular disorder wherein, in addition to administration of a smallmolecule that blocks or ameliorates or otherwise antagonizes or inhibitshemichannel opening alone or together with a small molecule that blocksor ameliorates or otherwise antagonizes or inhibits pannexin channelopening, the method also includes administering to the patient ananti-VEGF agent and/or an anti-PDGF agent as a therapeutic treatment. Inone aspect, a PDGF antagonist and/or a VEGF antagonist is/areadministered to a patient simultaneously with, or within about 1 to 5,10 or 90 days of, administration of, a hemichannel antagonist and/orpannexin channel antagonist, in amounts sufficient to suppress theneovascular disorder in the patient. In a particular embodiment of themethod of the invention, the PDGF antagonist and/or the VEGF antagonistare administered simultaneously with the hemichannel antagonist orinhibitor and/or the pannexin channel antagonist or inhibitor. In oneembodiment, the PDGF antagonist is a PDGF-B antagonist. In anotherembodiment, the VEGF antagonist is a VEGF-A antagonist. In certainembodiments, the PDGF antagonist is a nucleic acid molecule, an aptamer,an antisense RNA molecule, a ribozyme, an RNAi molecule, a protein, apeptide, a cyclic peptide, an antibody, a binding fragment of anantibody fragment, a sugar, a polymer, or a small molecule. In anotherembodiment, the VEGF antagonist is a nucleic acid molecule, an aptamer,an antisense RNA molecule, a ribozyme, an RNAi molecule, a protein, apeptide, a cyclic peptide, an antibody, a binding fragment of anantibody fragment, a sugar, a polymer, or a small molecule. In aparticular embodiment, this method of the invention involvesadministration of a VEGF antagonist that is an aptamer, such as anEYE001 aptamer. In another embodiment, this method of the inventioninvolves administration of a VEGF antagonist that is an antibody orbinding fragment thereof, such as Avastin® (bevacizumab) or Lucentis®(ranibizumab). In a particular embodiment, this method of the inventioninvolves administration of a PDGF antagonist that is an aptamer, anantibody or a binding fragment thereof. In another particularembodiment, this method of the invention involves administration of aPDGF antagonist that is an antisense oligonucleotide. In yet anotherembodiment of this aspect of the invention, the PDGF antagonist and/orthe VEGF antagonist are pro-drugs. In another embodiment, this method ofthe invention provides a means for suppressing or treating an ocularneovascular disorder. In some embodiments, ocular neovascular disordersamenable to treatment or suppression by the method of the inventioninclude ischemic retinopathy, iris neovascularization, intraocularneovascularization, age-related macular degeneration, cornealneovascularization, retinal neovascularization, choroidalneovascularization, diabetic retinal ischemia, or proliferative diabeticretinopathy. In still another embodiment, the method of the inventionprovides a means for suppressing or treating psoriasis or rheumatoidarthritis in a patient in need thereof or a patient diagnosed with or atrisk for developing such a disorder. The invention also provides apharmaceutical composition that includes a PDGF antagonist and/or a VEGFantagonist, as well a pharmaceutically acceptable carrier, along withone or more of a small molecule that blocks or ameliorates or otherwiseantagonizes or inhibits hemichannel opening alone or together with asmall molecule that blocks or ameliorates or otherwise antagonizes orinhibits pannexin channel opening. In this aspect, the PDGF and/or VEGFantagonists are present in amount(s) sufficient to suppress theneovascular disorder in the patient. This pharmaceutical composition ofthe invention may include a pharmaceutically acceptable carrier thatincludes a microsphere, nanoparticle, or a hydrogel formulation. Anotherembodiment of this aspect of the invention provides a pharmaceuticalpack that includes a PDGF antagonist and/or a VEGF antagonist togetherwith a small molecule that blocks or ameliorates or otherwiseantagonizes or inhibits hemichannel opening alone or together with asmall molecule that blocks or ameliorates or otherwise antagonizes orinhibits pannexin channel opening. In one embodiment of this aspect, thepharmaceutical pack includes a PDGF antagonist that is a PDGF-Bantagonist. In another embodiment of this aspect, the pharmaceuticalpack includes a VEGF antagonist that is a VEGF-A antagonist. In anotherembodiment, the PDGF antagonist and VEGF antagonist of thepharmaceutical pack are formulated separately and in individual dosageamounts. In still another embodiment, the PDGF antagonist and VEGFantagonist of the pharmaceutical pack are formulated together. It willbe seen that the combination of an anti-VEGF agent and/or an anti-PDGFagent can also afford surprisingly synergistic therapeutic benefits fortreating an ocular neovascular disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-FIG. 1F. Scrape loading studies using Lucifer yellow spread toneighbouring hCMVEC cells as an indication of gap junction couplinglevels. In the no treatment arm dye spreads readily to neighbouringcells (FIG. 1A). Peptide5 at the lower dose of 100 μM has little effecton coupling (FIG. 1B) but at the higher 500 μM dose there is animmediate reduction in spread (FIG. 1C). Over time, both peptideconcentrations show gap junction uncoupling by two hours (FIG. 1D andFIG. 1E). The graph to the right shows quantification of a similarexperiment comparing peptide5 blocking efficacy (immediate block, 500μM) with the non-specific gap junction channel blocker carbenoxolone(FIG. 1F).

FIG. 2. Quantification of scrape loaded dye spread in hCMVEC cellstreated with 50 μM Tonabersat for various periods of time. Tonabersathas some immediate channel blocking effect, and its efficacy increaseswith time. *=p<0.05, **=p<0.001, ***=p<0.001.

FIG. 3A-FIG. 3C. Tonabersat knockdown of Cx43 in APRE-19 cells in a dosedependent manner. ARPE-19 cells in control medium (FIG. 3A) and after 6hours preincubation with Tonabersat at 50 (FIG. 3B) and 100 (FIG. 3C)micromolar concentrations. Cells have been labelled for Connexin43. Incontrol cells the gap junctions are clearly localized at cell-cellinterfaces but after 6 hours with 50 micromolar Tonabersat mostjunctions have been internalised or lost. In the higher 100 micromolarTonabersat for 6 hours very little Connexin43 labelling remains.

FIG. 4A-FIG. 4B1. DAPI (nuclear staining) and labelling of the gapjunction protein Connexin43 (left panels, FIG. 4A and FIG. 4B) andmatching Connexin43 only labelling (right hand panels, FIG. 4A1 and FIG.4B1) in ARPE-19 cells. In untreated cells gap junction labelling isprimarily at cell-cell interfaces (top panels, FIG. 4A and FIG. 4A1). Incells treated for one hour with peptide5 (500 μM) the cell-cellinterfaces are still apparent but the bulk of the gap junctions havebeen internalized (lower panels (FIG. 4B and FIG. 4B1).

FIG. 5A-FIG. 5B. Electrophysiological recordings in Connexin43transfected HeLa cells (top traces, FIG. 5A) and connexin null HeLacells (bottom traces, FIG. 5B). As the voltage steps are raised,increased channel activity is observed in the transfected cells but notpresent in the connexin null cells. This channel activity in thetransfected cells is therefore the result of Connexin43 hemichannelopening.

FIG. 6A-FIG. 6C. Electrophysiological traces from Connexin43 transfectedHeLa cells that have been treated with the non-specific channel blockerscarbenoxolone (FIG. 6B) and LaC13 (FIG. 6A), and with peptide5 (100 μM)(FIG. 6C). The non-specific channel blockers show virtually completehemichannel block (compared with untreated HeLa cells in FIG. 7A-7C)with little channel activity remaining. Peptide5 also shows significanthemichannel blockade.

FIG. 7A-FIG. 7C. Electrophysiological traces form an equivalentexperiment to those shown in FIGS. 5A-5B and FIGS. 6A-6C. In this caseHCMVEC cells are shown prior to addition of Tonabersat (50 μM) (toppanel, FIG. 7A), during incubation with Tonabersat (middle panel, FIG.7B), and after three minutes of washout (bottom panel, FIG. 7C). Theresults demonstrate that Tonabersat almost completely abolisheshemichannel activity, with signs of activity returning after washout,indicating that Tonabersat is a very effective and direct hemichannelblocker.

FIG. 8A-FIG. 8C. Tonabersat inhibits hemichannel-mediated ATP releasefrom injured hCMVEC cells. (FIG. 8A) ATP released from hCMVEC cellsfollowing 2-hour exposure to injury in vitro. Quantification of totalextracellular ATP release is presented as a percentage of the injurycontrol for each treatment group. A significant reduction in ATP waspresent across all treatment groups compared to injury control: 100 μMCBX, 100 μM Peptide5 and 1 mM Probenecid, and 100 μM Peptide5 incombination with 1 mM Probenecid; the latter combination reduced totalATP release to the same level as CBX. (FIG. 8B) Quantification of totalextracellular ATP release is presented as a percentage of the injurycontrol for each treatment group. A significant reduction in ATP waspresent across all treatment groups compared to injury control: 100 μMCBX, 0.1 μM to 100 μM Tonabersat in combination with 1 mM Probenecid.(FIG. 8C) In this experiment, Tonabersat did not reduce ATP release fromhCMVEC cells in the absence of 1 mM Probenecid (p>0.09) One-way ANOVA,Tukey's multiple comparison test. Values represent mean±standard error.***P<0.001 against injury control.

FIG. 9. Hypoxia Reperfusion: ATP released from sub-confluent hCMVECcells following 2-hour exposure to injury then 2-hour exposure toreperfusion in vitro. ATP is quantified as a percentage of theinjury-reperfusion (IR) control. A significant reduction in ATP wasobserved with 100 μM CBX, 100 μM Peptide5, and 100 μM Peptide5 and 1 mMProbenecid, but not with 1 mM Probenecid on its own. A significantreduction in ATP was also observed with 10 μM Tonabersat. Valuesrepresent mean±standard error. One-way ANOVA Tukey's multiple comparisontest *P<0.05 against IR control.

FIG. 10A-FIG. 10D. 1 hour treatment with Tonabersat down-regulates Cx43GJ plaques via the lysosomal degradation pathway in ARPE-19 cells. (FIG.10A and FIG. 10B) Immunolabeling of Cx43 in 100 μM Tonabersat (above,FIG. 10A), and 100 μM Tonabersat+NH4Cl for 1 h in serum-supplementedmedia (n=3 wells, 2 independent experiments) (FIG. 10B) Tonabersatcontrols (FIG. 10C) Quantification of the total area of Cx43 plaques percell following treatment with Tonabersat (5-500 μM) for 1 h. (FIG. 10D)Quantification of the total area of Cx43 plaques per cell followingtreatment with Tonabersat (50-500 μM) with NH4Cl for 6 h (D). (FIG. 10C)and (FIG. 10D) are normalized to untreated control. Significantdifferences are represented as ***p<0.0001. Bars represent mean±S.E.Mand the differences between. Scale bars=30 μm.

FIG. 11A-FIG. 11C. Tonabersat internalises and down regulates Cx43 GJplaques in ARPE-19 cells. (FIG. 11A) Immunolabeling of Cx43 in untreatedcontrol (above), 100 μM Tonabersat (below), for 1 h inserum-supplemented media (n=3 wells, 2 independent experiments) (FIG.11B) Quantification of the total area of Cx43 plaques per cell followingtreatment with Tonabersat (5-500 μM) for 1 h. (FIG. 11C) Quantificationof the total area of Cx43 plaques per cell following treatment withTonabersat (5-500 μM) for 6 h. B and C are normalized to untreatedcontrol. Bars represent mean±S.E.M and the significant differencerepresent ***p<0.0001. Statistical test One-way ANOVA, Scale bars=30 μm.

FIG. 12A-FIG. 12C. 6 h treatment with Tonabersat downregulates Cx43 GJplaques via the lysosomal degradation pathway in ARPE-19 cells.Immunolabeling of Cx43 in 100 μM Tonabersat (above, FIG. 12A), and 100μM Tonabersat+NH4Cl for 6 h (FIG. 12A1) in serum-supplemented media (n=3wells, 2 independent experiments) (FIG. 12B) Quantification of the totalarea of Cx43 plaques per cell following treatment with Tonabersat (5-500μM) for 1 h. (FIG. 12C) Quantification of the total area of Cx43 plaquesper cell following treatment with Tonabersat (50-500 μM) with NH4Cl for6 h. (FIG. 12B) and (FIG. 12C) are normalized to untreated control.Significant difference ***p<0.0001. Bars represent mean±S.E.M and thedifferences between. Scale bars=30 μm.

FIG. 13. Cx43 mRNA levels in confluent monolayers of ARPE-19 cellsincubated with DMSO (vehicle control), or with 50 μM Tonabersat for 1 h.Bars represent means±S.E.M (n=3). There was no significant difference inCx43 mRNA in ARPE-19 cells following treatment with 50 μM tonabersatcompared to both untreated (p=0.7572) and vehicle controls (p=0.10245).One-way ANOVA Tukey's multiple-comparisons test.

FIG. 14A-FIG. 14B. Comparison of (FIG. 14A) ATP concentration, or (FIG.14B) % Cell Viability, relative to control, for the various treatmentgroups.

FIG. 15. Retinal ganglion cell density 7 days after retinal ischemia at120 mmHg for 60 minutes. Results for both the ischaemic eyes andcontralateral eyes are shown. Mean±standard error. N=2, 6, 6, 6, 5, 4,4. NT, no treatment; T, Tonabersat. A statistically significant (p<0.01)31% loss of RGCs was found in ischaemic eyes with no treatment(167111±14188 cells/cm2, mean±standard error) compared to normalcontrols (242558±15840 cells/cm2). A statistically significant (p=0.03)35% RGC loss was found in ischaemic eyes treated with 10 mg/kgTonabersat (158739±26370 cells/cm2). However, ischaemic eyes treatedwith 1 mg/kg tonabersat sustained fewer cell loss (18%, 199927±26058cells/cm2) than the other groups. The difference between this group andthe normal controls was a trend (p=0.17). No loss of RGCs was found inall contralateral eyes.

FIG. 16A1-FIG. 16D. Functional effect of Tonabersat on intercellularcommunication in hCMVEC cells in vitro. (FIG. 16A1, FIG. 16A2) Invehicle control, LY dye spreads readily to neighbouring cells viacoupled GJs. Immediate (FIG. 16A3, FIG. 16A4) or 2-hour (FIG. 16A5, FIG.16A6) Tonabersat treatment (50 μM) causes a reduction in LY dyetransfer, as GJs are uncoupled. (FIG. 16B) Quantification of LY positivehCMVEC cells treated with 50 μM Tonabersat at immediate to 24 htime-intervals. Tonabersat-mediated uncoupling of GJs increases withtime. (FIG. 16C) Lower concentrations of Tonabersat (10 μM, 1 μM, 0.1μM) did not cause GJ uncoupling after two hours in this experiment, orafter (FIG. 16D) 24 hours. Bars represent mean±S.E.M (One-way ANOVAfollowed by Tukey's post-hoc test) n=3 wells, 3 independent experiments.*=p<0.05, **=p<0.001, ***=p<0.001.

FIG. 17A-FIG. 17C. FACS data for the transfection uptake of severalselected antisense oligonucleotides. (FIG. 17A) NT, (FIG. 17B) SEQ4-O,(FIG. 17C.) SEQ4-PTO.

FIG. 18. Image of live cells via confocal microscopy shows uptake ofFAM-labelled SEQ4-PTO (modified) in HUVEC cells (4 hrpost-transfection). Green represents the oligo SEQ1-O (FAM labelled),Blue represents cell nucleus stained by DAPI.

FIG. 19. Green represents the oligo SEQ1-O (FAM labelled), Bluerepresents cell nucleus stained by DAPI.

FIG. 20. Shows the knockdown efficiency of Connexin 43 as measured byqPCR. The data shows the standard deviation for 3 replicates.

FIG. 21. Knockdown efficiency of Connexin 43 as measured by Western Blot(n=3). The data show standard deviation for 3 replicates.

FIG. 22A-FIG. 22B Knockdown efficiency of modified (PTO) and unmodifiedsequences measured by qPCR at 4 hours (FIG. 22A) and 8 hours (FIG. 22B)after transfection, respectively. Oligo concentration: (1)=200 nM;(3/4)=150 nM; (1/2)=100 nM; C: SEQ1; SEQ4: 37501; Cscr: SEQ1 scrambled;LP2scr: SEQ4 scrambled; Orange bars: negative controls. (n=3 for eachresult).

FIG. 23A-FIG. 23B. (FIG. 23A) and (FIG. 23B) Knockdown efficiency asmeasured by Western blot. The data show standard deviation for 3replicates. Knockdown efficiency as measured by Western blot (n=3).Oligo concentration: (1)=200 nM; (3/4)=150 nM; (1/2)=100 nM; C: SEQ1;SEQ4: 37501; Cscr: SEQ1 scrambled; LP2scr: SEQ4 scrambled; Orange bars:negative controls

FIG. 24. Different sequences analyzed by qPCR for their knockdownefficiency of connexin 43. Cscr: SEQ1 scrambled, Csen: SEQ1 sensestrand, LP2scr2: SEQ4 scrambled, LP2sen: SEQ4 sense strand, 47001scr2:SEQ5 scrambled, 47001sen: SEQ5 sense strand, NC1: universal negative 1,NC2: universal negative 2

FIG. 25. Different sequences analyzed by Western Blot for theirknockdown efficiency of connexin 43. Cscr: SEQ1 scrambled, Csen: SEQ1sense strand, LP2scr2: SEQ4 scrambled, LP2sen: SEQ4 sense strand,47001scr2: SEQ5 scrambled, 47001sen: SEQ5 sense strand, NC1: universalnegative 1, NC2: universal negative 2.

FIG. 26. Cxn43 knockdown results of oligonucleotide sequences indifferent cell types. H: human, P: pig, LP2: SEQ4, C: SEQ1, Cscr: SEQ1scrambled, Csen: SEQ1 sense strand, 47001scr2: SEQ5 scrambled, 47001sen:SEQ5 sense strand, the other numbers represent other antisense sequencesto Connexin 43, as described herein.

FIG. 27. Dose-response performance in HUVEC cells by qPCR for variousoligonucleotide sequences. LP2: SEQ4, Coda: SEQ1, 47001scr2: SEQ5scrambled, the other numbers represent other antisense sequences toConnexin 43, as described herein.

FIG. 28A and FIG. 28B Knockdown efficiency of Cxn43 by the unmodifiedsequences (no phosphorthioate linkages in the backbone) as measured byqPCR. Oligo concentration: (1)=200 nM; (2)=400 nM; (3)=600 nM C: SEQ1;SEQ4: 37501; Cscr: SEQ1 scrambled; LP2scr: SEQ4 scrambled.

FIG. 29A and FIG. 29B Knockdown efficiency comparison of the modified(with all-thiophosphoriate backbone) sequences. Oligo concentration:(1)=200 nM; (2)=400 nM; (3)=600 nM C: SEQ1; SEQ4: 37501; Cscr: SEQ1scrambled; LP2scr: SEQ4 scrambled

FIG. 30A and FIG. 30B In vitro knockdown of unmodified oligo sequences.Cscr: SEQ1 scrambled; 133704: another antisense oligo to Connexin 43.

FIG. 31 Comparative Knockdown activity of SEQ ID NO.: 1, SEQ ID NO.:4,and the ASN from Table 58 at 4 hr post-transfection.

FIG. 32. Comparative Knockdown activity of SEQ ID NO.: 1, SEQ ID NO.:4,and the ASN from Table 58 at 12 hr post-transfection.

FIG. 33. Dose-response curves for candidate Connexin 43 antisenseoligonucleotides.

FIG. 34A-FIG. 34D. Dye perfusion of Evans Blue Dye post-ischemia to mapthe connexin 43 following ischemia-reperfusion. (FIG. 34A) Controlretina—no dye perfusion. (FIG. 34B) Control retina—Evans Blue Dyeintraperitineal injection. (FIG. 34C) Dye injection 4 hourspost-ischemia. (FIG. 34D) Dye injection 24 hours post-ischemia.

FIG. 35A-FIG. 35B. The calculated (via ImageJ, as described herein) areaof dye leak (FIG. 35A) or Connexin 43 spot count (FIG. 35B) as afunction of time following ischemia-reperfusion. The results show thebaseline performance of dye leakage.

FIG. 36. The effects on the total dye leak following treatment with, andwithout, the Cxn43 modulating agent.

FIG. 37. The ability to retard the expression of Cxn43 (Y-axis) comparedfor variants of a particular core sequence. Peptide 5=SEQ ID NO: 168,Mod 1=SEQ ID NO: 171, Mod 2=SEQ ID NO: 172, Mod 3=SEQ ID NO: 173, Mod4=SEQ ID NO: 174, Mod 5=SEQ ID NO: 175, Mod 6=SEQ ID NO: 176.

FIG. 38A-FIG. 38E. SEM images of nanoparticles (Nps, FIG. 38A and FIG.38C) and Microparticles (Mps, FIG. 38B and FIG. 38D), before (FIG. 38Aand FIG. 38B) and after (FIG. 38C and FIG. 38D) three days in releasemedia. FIG. 38E shows formulation, particle size, PDI, ZP (mV), Yield(%), and EE (%) of Mps and Nps.

FIG. 39. In vitro release study of connexin 43 modulator fromparticulate formulations (data points represent mean values ±SD, n=3).

FIG. 40A-FIG. 40E. Measurement of Vessel leak, post-ischemia, ofunmodified (Cxn43 MP) and chemically modified (C12-C12 Cxn43 MP)connexin 43 modulator peptides. Vessel leak (4 hours post ischemia):(FIG. 40A) Control, (FIG. 40B) Ischaemia only, (FIG. 40C) Cx43 MP, (FIG.40D) C₁₂-C₁₂-Cx43 MP. (FIG. 40E) Area of vessel leak.

FIG. 41A-FIG. 41D. Representative images of stained tissues andquantitation of spot counts which were reduced moreso with thechemically modified (C12-C12 Cxn43 MP) connexin 43 modulator peptidethan the unmodified peptide. Cx43 spot counts (wholemounts): (FIG. 41A)Control, (FIG. 41B) Ischaemia 8 h, (FIG. 41C) 2C12-Cx43 MP 8 h. (FIG.41D) Density Cx43 spots/mm².

FIG. 42A-FIG. 42D. Representative images of stained tissues andquantitation that formulations involving nanoparticles for the deliveryresulted in lower Cxn43 expression than formulations without thenanoparticles. Cx43 spot counts 28 days: (FIG. 42A) Control, (FIG. 42B)Ischaemia 28d, (FIG. 42C) 2C₁₂-Cx43 MP 28d. (FIG. 42D) Mean Cx43 count.

FIG. 43A-FIG. 43D. Representative images of stained tissues andquantitation that the chemically modified peptide treatment resulted inover 93% RGC survival after 28 days compare to less than 70% foruntreated eyes (ischemia only). Retinal ganglion cell count: (FIG. 43A)Control, (FIG. 43B) Ischaemia 28D, (FIG. 43C) 2C12-Cx43MP 28D. (FIG.43D) Density cells/mm².

FIG. 44A-FIG. 44B. Representative images of stained tissues indicatingCxn43 distribution located within the choroid of a young (29 year old)donor without AMD (FIG. 44A), and an elderly donor diagnosed with AMD(FIG. 44B). In the young donor the Cxn43 labeling (red, under the “RPE”label) is especially dense adjacent to Bruch's membrane, and the labelprimarily demarcates gap junctions between endothelial cells (whitearrow). The elongated nuclei stained with the nuclear marker DAPI areendothelial cell nuclei. Scale bars represent 20 μm.

FIG. 45. Representative images of stained tissues indicating Cxn43expression profile in retina images taken near a haemorrhage of a retinasample of a deceased human. Merged images of nuclei stained with DAPI(blue) and Cxn43 gap junction plaques (red).

FIG. 46. Representative images of stained tissues indicating Cxn43expression profile in retina images taken near a haemorrhage of a retinasample of a deceased human. The figure shows merged images of nucleistained with DAPI (blue) and Cxn43 gap junction plaques (red).

FIG. 47A-FIG. 47G. Representative confocal microscope images of retinasindicating the Cxn43 expression profile in ARPE-19 cells. Control (FIG.47A). After 8 h of exposure to the native peptide (FIG. 47B), Cxn43levels in ARPE-19 cells were slightly reduced while after 24 h (FIG.47C), Cxn43 was back to normal (FIG. 47A). There were no significantdifferences in Cxn43 labelling after 8 h of incubation with Nps (FIG.47D) and Mps (FIG. 47F), while after 24 h of exposure, both Nps (FIG.47E) and Mps (FIG. 47G) groups exhibited a considerable reduction inCxn43 levels indicating sustained peptide release and thus the potentialof longer term treatment with these particles possibly reducing new gapjunction channel formation. The figure shows merged images of nucleistained with DAPI (blue) and Cxn43 gap junction plaques (red) (Scale barrepresents 50 μm).

FIG. 48A-FIG. 48G. Representative confocal microscope images of retinaslabelled for GFAP (red), Cxn43 (green) and DAPI (blue). Control (FIG.48A). Twenty-eight days after ischemia-reperfusion, Cx43 and GFAP weresignificantly upregulated in the untreated group (FIG. 48B).Intravitreal injection of native Cxn43 MP in solution displayed limitedCxn43 upregulation after 28 d (FIG. 48C). The Nps-Cxn43 MP treatmentgroup resulted in a significant reduction of Cxn43 upregulation at 28 dpost-injury (FIG. 48D), while Mps-Cxn43 MP exhibited similar Cxn43 andGFAP levels at 28 d (FIG. 48E) and 90 d (FIG. 48F) followingischemia-reperfusion (scale bar represents 50 μm). (FIG. 48G) AverageCxn43 spot counts in uninjured control retinas and followingischemia-reperfusion without and with treatment. Stars denotestatistical significance between each group (n=3, mean±SD, *p<0.05,**p<0.01).

FIG. 49A-FIG. 49H. Representative confocal microscope images of flatmounted retinas with Brn3a labelled RGC (red) post ischemia-reperfusion.Control (FIG. 49A). RGC distribution is significantly reduced withalmost complete loss of blood vessel delineation in untreated retinas(FIG. 49B). Eyes treated with Cxn43 MP in solution and Nps-Cxn43 MPexhibited fewer patches of RGC loss (FIG. 49C and FIG. 49D). Eyestreated with Mps still exhibited some RGC loss at 28 d (FIG. 49E) and 90d (FIG. 49E). Scale bar=300 lm. (FIG. 49G) Average density of RGC. (FIG.49H) Stars denote statistical significance between each group (n=6,mean±SD, **p<0.01, *p<0.05).

DETAILED DISCLOSURE

The inventions relate to modulators of gap junction channels, modulatorsof hemichannels, modulators of pannexin channels, modulators of pannexintranscription, translation, function and/or activity, and modulators ofconnexin transcription, translation, function and/or activity, includingsmall molecule modulators. Modulators may be used alone or incombination to treat a disease, disorder or condition as describedherein, including ocular diseases, disorders or conditions in theanterior segment, the posterior segment, and in ocular blood vesselsincluding in the retina, the choroid and the choriocapillaris.

Tonabersat, a benzoylamino benzopyran, has been reported to be centralnervous system-specific and to act at the level of downregulatingconnexin 26 and/or p38 expression. The inventors have surprisinglyidentified that gap junction channel modulators such as Tonabersat canmodulate the activity of gap junction channels and hemichannels in anumber of different cell types which are not limited to the centralnervous system and in a manner which is not specific to a particularconnexin (such as connexin 26). In particular, the inventors have shownthat Tonabersat can modulate the activity of hemichannels comprisingconnexin 43. While not wishing to be bound by any particular theory, theinventors have surprisingly discovered that tonabersat acts directly ongap junction channels and/or hemichannels, rather than indirectlythrough a cellular receptor or by affecting the expression of a connexin(for example, connexin 26) and/or p38.

It has been shown that a gap junction channel modulator such as peptide5 inhibits Cx43 hemichannel activity and/or ATP release during andfollowing injury, for example, during ischemia and in hypoxiareperfusion, as described in detail herein. It has also been shown thata pannexin modulator such as Probenecid inhibits injury induced ATPrelease, for example, ATP release during ischemia.

The instant inventions provides, inter alia, (1) methods for modulationof a gap junction channel and/or hemichannel; (2) the use of a gapjunction channel modulator, such as peptide 5, and/or an analoguethereof, compounds of formula I, for example tonabersat, and/or ananalogue or pro-drug of any of the foregoing compounds, and/or apannexin modulator, e.g., probenecid and/or a synthetic mimetic peptideblocker of pannexin 1, e.g., ¹⁰Panx1, or an analogue of either thereof,in the manufacture of a medicament for modulation of a gap junctionand/or hemichannel; (3) a gap junction channel modulator, such aspeptide 5, and/or an analogue thereof, compounds of formula I, forexample tonabersat, and/or an analogue or pro-drug of any of theforegoing compounds, and/or, a pannexin modulator, e.g., probenecidand/or a synthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1, or an analogue of either thereof, for modulating a gap junctionchannel and/or hemichannel; (4) methods for the treatment of a disorderwhere modulation of a gap junction channel, hemichannel, and/or pannexinchannel may be of benefit by administering a gap junction channelmodulator, such as peptide 5, and/or an analogue thereof, compounds offormula I, for example tonabersat, and/or an analogue or pro-drug of anyof the foregoing compounds, and/or, a pannexin modulator, e.g.,probenecid and/or a synthetic mimetic peptide blocker of pannexin 1,e.g., ¹⁰Panx1, or an analogue of either thereof; (5) the use of a gapjunction channel modulator, such as peptide 5, peptide 5, and/or ananalogue thereof, compounds of formula I, for example tonabersat, and/oran analogue or pro-drug of any of the foregoing compounds, and/or, apannexin modulator, e.g., probenecid and/or a synthetic mimetic peptideblocker of pannexin 1, e.g., ¹⁰Panx1, or an analogue of either thereof,in the manufacture of a medicament for the treatment of a disorder wheremodulation of a gap junction channel, hemichannel, and/or pannexinchannel may be of benefit; and (6) a gap junction channel modulator,such as peptide 5, and/or an analogue thereof, compounds of formula I,for example tonabersat, and/or an analogue or pro-drug of any of theforegoing compounds, and/or, a pannexin modulator, e.g., probenecidand/or a synthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰panx1,or an analogue of either thereof, for use in the treatment of a disorderwhere modulation of a gap junction channel and/or hemichannel may be ofbenefit.

In some embodiments, this invention relates to the use of a pannexinmodulator, e.g., probenecid and/or a synthetic mimetic peptide blockerof pannexin 1, e.g., ¹⁰Panx1, or an analogue of either thereof, for oneof more of these purposes, either alone or in combination with a gapjunction channel modulator of this disclosure.

In certain embodiments, the inventors contemplate the inventionproviding one or more of the following methods which comprisingadministering a gap junction channel modulator such as peptide 5, and/oran analogue thereof, compounds of formula I, for example tonabersat,and/or an analogue or pro-drug of any of the foregoing compounds, and/oradministering a pannexin modulator, e.g., probenecid and/or a syntheticmimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1, or an analogue ofeither thereof, to a subject, either alone or in combination with a gapjunction channel modulator: a method for preventing or decreasingabnormal or excessive scar formation in a subject undergoing a surgicalprocedure; a method for treating a subject having abnormal scarring byexcising the scar and then administering a gap junction channelmodulator such as peptide 5, and/or an analogue thereof, compounds offormula I, for example tonabersat, and/or an analogue or pro-drug of anyof the foregoing compounds, and/or administering a pannexin modulator,e.g., probenecid and/or a synthetic mimetic peptide blocker of pannexin1, e.g., ¹⁰Panx1, or an analogue of either thereof, to the subject atthe site of excision, either alone or in combination with a gap junctionchannel modulator; the use of a gap junction channel modulator such aspeptide 5, and/or an analogue thereof, compounds of formula I, forexample tonabersat, and/or an analogue or pro-drug of any of theforegoing compounds, and/or administering a pannexin modulator, e.g.,probenecid and/or a synthetic mimetic peptide blocker of pannexin 1,e.g., ¹⁰Panx 1, or an analogue of either thereof, either alone or incombination with a gap junction channel modulator in any of thefollowing methods: a method of preventing or decreasing contracture in atissue of a subject and thereby decreasing fibrosis; a method for tissueengineering in association with an ophthalmic procedure; a method ofpromoting the accumulation of epithelial cells in the eye or in a tissueassociated with the eye; a method of inhibiting hypercellularity in theeye or in a tissue associated with the eye; a method for preventing,decreasing or lessening lesion spread; a method for the treatment ofvascular leak or any disorder in which vascular leak is implicated; amethod for the treatment of one or more of ischemia (including forexample, perinatal ischemia, skin ischemia and cardiac ischemia), brainstroke, asphyxia, brain trauma, spinal cord injury, heart attack,inflammatory cardiac insult (including for example, pericarditis),reperfusion injury (including for example, cardiac reperfusion aftersurgery or transplant, liver reperfusion after surgery or transplant); amethod for treating injury, tissue damage or inflammation associatedwith a surgical procedure or medical treatment (including for example,chemotherapy, radiotherapy, plastic surgery, and for example includingstomatitis or rash associated with such treatments); a method for thetreatment of surface wounds or lesions (including for example,persistent epithelial defects in the cornea, ulcers, burns, psoriasis);a method for the treatment of a hearing disorder; a method for thetreatment of an ocular disorder (including for example, retinal vein orartery occlusion, glaucoma, retinal stroke, trauma resulting in raisedintraocular pressure, diabetic retinopathy, cystoid macular edema, agerelated macular degeneration, infection, burns (including chemical andthermal, for example)); a method for the treatment of epilepsy,Parkinson's disease; and, a method for the treatment of trauma(including trauma which is associated with blood vessel haemorrhage,edema, lesions, lesion spread, and/or inflammation, for example). Inaddition, the inventors contemplate the invention providing: a dressingfor preventing and/or treating fibrosis or other disorder comprising agap junction channel modulator such as peptide 5, and/or an analoguethereof, compounds of formula I, for example tonabersat, and/or ananalogue or pro-drug of any of the foregoing compounds, and/or apannexin modulator, e.g., probenecid and/or a synthetic mimetic peptideblocker of pannexin 1, e.g., ¹⁰Panx1, or an analogue of either thereof;and/or, the use of a gap junction channel modulator such as peptide 5,and/or an analogue thereof, compounds of formula I, for exampletonabersat, and/or an analogue or pro-drug of any of the foregoingcompounds, and/or administering a pannexin modulator, e.g., probenecidand/or a synthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1, or an analogue of either thereof, in the manufacture of a medicamentfor the treatment of a disorder as herein described.

In some embodiments, this invention features the use of compounds offormula I, for example tonabersat, and/or an analogue or pro-drug of anyof the foregoing compounds to directly and immediately block Cx43hemichannels and to cause a concentration and time-dependent reductionin GJ coupling. In some aspects a low concentration of compounds offormula I, for example tonabersat, and/or an analogue or pro-drug of anyof the foregoing compounds can be used in any of the methods oftreatment or uses of this invention. In some embodiments, a low dose ofcompounds of formula I, for example tonabersat, and/or an analogue orpro-drug of any of the foregoing compounds may be combined with apannexin channel modulator in any of the methods of treatment or uses ofthis invention. In some embodiments, a low dose of compounds of formulaI, for example tonabersat, and/or an analogue or pro-drug of any of theforegoing compounds may be combined with a pannexin channel modulatormay be used, for example, as an effective treatment during ischaemicinjury, to protect RGCs from damage following retinal ischemia, or totreat retinal ischemia or eye fibrosis, or in any of the other methodsof treatment or uses of this invention.

Channel Modulators

By way of example, tonabersat may be known by the IUPAC nameN-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrochromen-4-yl]-3-chloro-4-fluorobenzamideor(3S-cis)-N-(6-acetyl-3,4-dihydro-3-hydroxy-2,2-(dimethyl-d6)-2H-1-benzopyran-4-yl)-3-chloro-4-fluorobenzamide.

In one embodiment, tonabersat and/or an analogue or prodrug thereof ischosen from the group of compounds having the formula I:

Y is C—R₁;

R₁ is acetyl;R₂ is hydrogen, C₃₋₈ cycloalkyl, C₁₋₆ alkyl optionally interrupted byoxygen or substituted by hydroxy, C₁₋₆ alkoxy or substitutedaminocarbonyl, C₁₋₆ alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆alkylcarbonyloxy, C₁₋₆ alkoxy, nitro, cyano, halo, trifluoromethyl, orCF₃S; or a group CF₃-A-, where A is —CF₂—, —CO—, —CH₂—, CH(OH), SO₂, SO,CH₂—O—, or CONH; or a group CF₂H-A′- where A′ is oxygen, sulphur, SO,SO₂, CF₂ or CFH; trifluoromethoxy, C₁₋₆ alkylsulphinyl, perfluoro C₂₋₆alkylsulphonyl, C₁₋₆ alkylsulphonyl, C₁₋₆ alkoxysulphinyl, C₁₋₆alkoxysulphonyl, aryl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,phosphono, arylcarbonyloxy, heteroarylcarbonyloxy, arylsulphinyl,heteroarylsulphinyl, arylsulphonyl, or heteroarylsulphonyl in which anyaromatic moiety is optionally substituted, C₁₋₆ alkylcarbonylamino, C₁₋₆alkoxycarbonylamino, C₁₋₆ alkyl-thiocarbonyl, C₁₋₆ alkoxy-thiocarbonyl,C₁₋₆ alkyl-thiocarbonyloxy, 1-mercapto C₂₋₇ alkyl, formyl, oraminosulphinyl, aminosulphonyl or aminocarbonyl, in which any aminomoiety is optionally substituted by one or two C₁₋₆ alkyl groups, orC₁₋₆ alkylsulphinylamino, C₁₋₆ alkylsulphonylamino, C₁₋₆alkoxysulphinylamino or C₁₋₆ alkoxysulphonylamino, or ethylenylterminally substituted by C₁₋₆ alkylcarbonyl, nitro or cyano, or —C(C₁₋₆alkyl)NOH or —C(C₁₋₆ alkyl)NNH₂; or amino optionally substituted by oneor two C₁₋₆ alkyl or by C₂₋₇ alkanoyl; one of R₃ and R₄ is hydrogen orC₁₋₄ alkyl and the other is C₁₋₄ alkyl, CF₃ or CH₂X^(a) is fluoro,chloro, bromo, iodo, C₁₋₄ alkoxy, hydroxy, C₁₋₄ alkylcarbonyloxy, —S—C₁₋₄ alkyl, nitro, amino optionally substituted by one or two C₁₋₄ alkylgroups, cyano or C₁₋₄ alkoxycarbonyl; or R₃ and R₄ together are C₂₋₅polymethylene optionally substituted by C₁₋₄ alkyl;

R₅ is C₁₋₆ alkylcarbonyloxy, benzoyloxy, ONO₂, benzyloxy, phenyloxy orC₁₋₆ alkoxy and R₆ and R₉ are hydrogen or R₅ is hydroxy and R₆ ishydrogen or C₁₋₂ alkyl and R₉ is hydrogen;

R₇ is heteroaryl or phenyl, both of which are optionally substituted oneor more times independently with a group or atom selected from chloro,fluoro, bromo, iodo, nitro, amino optionally substituted once or twiceby C₁₋₄ alkyl, cyano, azido, C₁₋₄ alkoxy, trifluoromethoxy andtrifluoromethyl;

R₈ is hydrogen, C₁₋₆ alkyl, OR₁₁ or NHCOR₁₀ wherein R₁₁ is hydrogen,C₁₋₆ alkyl, formyl, C₁₋₆ alkanoyl, aroyl or aryl-C₁₋₆ alkyl and R₁₀ ishydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, mono or di C₁₋₆ alkyl amino, amino,amino-C.sub.1-6 alkyl, hydroxy-C₁₋₆ alkyl, halo-C₁₋₆ alkyl, C₁₋₆acyloxy-C₁₋₆ alkyl, C₁₋₆ alkoxycarbonyl-C₁₋₆-alkyl, aryl or heteroaryl;the R₈—N—CO—R₇ group being cis to the R₅ group; and X is oxygen or NR₁₂where R₁₂ is hydrogen or C₁₋₆ alkyl.

For any of the Markush groups set forth above, that group can include orexclude any of the species listed for that group.

Tonabersat may be known by the IUPAC nameN-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrochromen-4-yl]-3-chloro-4-fluorobenzamideor(3S-cis)-N-(6-acetyl-3,4-dihydro-3-hydroxy-2,2-(dimethyl-d6)-2H-1-benzopyran-4-yl)-3-chloro-4-fluorobenzamide.

In embodiment the analogue of formula 1 is the compound carabersat(N-[(3R,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrochromen-4-yl]-4-fluorobenzamide)ortrans-(+)-6-acetyl-4-(S)-(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-1-benzo[b]pyran-3R-ol,hemihydrate.

In certain embodiments, tonabersat and/or an analogue thereof are in theform of a free base or a pharmaceutically acceptable salt. By way ofexample, a pharmaceutically acceptable salt includes a hydrochloridesalt and salts derived from acid including, but not limited to,1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid,2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoicacid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid(L), aspartic acid (L), benzenesulfonic acid (besylate), benzoic acid,camphoric acid (+), camphor-10-sulfonic acid (+), capric acid (decanoicacid), caproic acid (hexanoic acid), caprylic acid (octanoic acid),carbonic acid, cinnamic acid, citric acid, cyclamic acid,dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid,formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonicacid (D), gluconic acid (D), glucuronic acid (D), glutamic acid,glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid,hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid (DL),lactobionic acid, lauric acid, maleic acid, malic acid (−L), malonicacid, mandelic acid (DL), methanesulfonic acid (mesylate),naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinicacid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid,phosphoric acid, proprionic acid, pyroglutamic acid (−L), salicylicacid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaricacid (+L), thiocyanic acid, toluenesulfonic acid (p), and undecylenicacid. In one embodiment, the salt is a hydrochloride salt.

In other embodiments, one or more polymorph, one or more isomer, and/orone or more solvate of tonabersat and/or an analogue thereof may beused.

Peptide 5

Various small organic molecules have been reported to have activity ininhibition of gap junction or hemichannel currents. They includetriarylmethanes (TRAMS), quinine, mefloquine, fenamates,2-aminophenoxyborate and derivatives, glycyrrhetinic acid andderivatives, volatile anesthetics such as halothane and ethane,lipophilic compounds such as long-chain alcohols (e.g., heptanol andoctanol), fatty acid amides including oleamide, cyclodextrins,cisplatin, polyamines and tetraalylammonium ions. An increasing numberof studies have also reported on the inhibition of gap junction channelsand hemichannels using peptides corresponding to specific sequenceswithin extracellular loops E1 and E2 involving the conserved QPG andSHVR (SEQ ID NO: 18) motifs of E1 (Gap26 peptide) and the SRPTEK (SEQ IDNO: 20) motif in E2 (Gap27 peptide) as well as the cytoplasmic loop(Gap19 peptide). The most potent such peptidomimetic is Peptide5(VDCFLSRPTEKT) (SEQ. ID NO:168).

In some embodiments, the gap junction channel modulators are connexinpeptides or peptidomimetics, sometimes referred to anti-connexinpeptides or peptdomimetics, e.g., anti-connexin hemichannel blockingpeptides or peptidomimetics, for example, modified or unmodifiedpeptides or peptidomimentics comprising connexin extracellular domains,transmembrane regions, and connexin carboxy-terminal peptides). Theanti-connexin hemichannel blocking peptides or peptidomimetics may bemodified or unmodified. The anti-connexin hemichannel blocking peptidesor peptidomimetics are made chemically, synthetically, or otherwisemanufactured. In some embodiments, the gap junction channel modulatorsare connexin 43 peptides or peptidomimetics. In some aspects, thetherapeutically effective modified or unmodified peptide orpeptidomimetic comprises a portion of an extracellular or transmembranedomain of a connexin, such as connexin 43 or connexin 45. In someaspects peptide or peptidomimetic comprises a portion of anextracellular or transmembrane domain of connexin Cx26, Cx30, Cx31.1,Cx36, Cx37, Cx40, Cx50, Cx57 or any other connexin in the eye or bloodvessels.

In some embodiments the gap junction channel modulators of thisinvention include anti-connexin 43 peptides or peptidomimetics, forexample, any of the peptides described herein, including peptidescomprising a portion of an extracellular domain of a connexin, andpeptides comprising a portion of a carboxy-terminal portion of aconnexin useful in the methods of this invention, which istherapeutically effective, for example, effective for healing any of theneuropathic ocular disorders described herein. In some aspects, thetherapeutically effective modified or unmodified peptide orpeptidomimetic comprises a portion of an extracellular domain of aconnexin, such as connexin 43 or connexin 45, preferably connexin 43.

Peptide5 is an established gap junction channel blocker that can operatein a dose dependent manner, with lower doses blocking gap junctionhemichannel opening and higher doses uncoupling gap junctions betweencells. See, e.g., O'Carroll et al, 2008. With sustained low doseapplication of Peptide5 there is also gradual loss of gap junctioncoupling, considered to be peptide interference with hemichannel docking(in parallel with gradual removal of existing gap junctions duringnormal turnover). Peptide5 has proven to be effective in a number of invitro, ex vivo and in vivo (animal) studies, especially when used atdoses that block hemichannels without uncoupling gap junctions (see forexample Davidson et al, 2012; Danesh-Meyer et al, 2012; O'Carroll et al,2013). The results in O'Carroll et al, 2008 indicate that Peptide5 atlow or high concentration blocks hemichannels, but will uncouple gapjunctions directly at high concentrations. Peptide5 data is shown herefor comparison with tonabersat.

Compounds for pannexin modulation can be the compounds of formula VI:

wherein

Z₁ and Z₂ are independently selected from: (C₁-C₄)alkyl,halo(C₁-C₄)alkyl, (C₁-C₄)alkoxy, halo(C₁-C₄), (C₁-C₄)cycloalkyl,(C₁-C₄)alkyl-S—, (C₁-C₄)alkylamino, (C₁-C₄)cycloalkylamino,di(C₁-C₄)alkylamino, and amino(C₁-C₄)alkyl;

Y is selected from: hydrogen, halo, cyano, hydroxy, (C₁-C₄)alkyl,halo(C₁-C₄)alkyl, (C₁-C₄)alkoxy, halo(C₁-C₄)alkoxy, cyano(C₁-C₄)alkyl,amino, (C₁-C₄)alkylamino, di(C₁-C₄)alkylamino, amino(C₁-C₄)alkyl,(C₁-C₄)alkylamino(C₁-C₄)alkyl, di[(C₁-C₄)alkyl]amino(C₁-C₄)alkyl,trifluoromethylthio, hydroxy(C₁-C₄)alkyl, (C₁-C₄)alkoxy(C₁-C₄)alkyl,—C(O)R₁, —C(O)OR₁, —OC(O)R₁, —C(O)—N(R₁)₂, —CH₂—C(O)R₁, —CH₂—C(O)OR₁,—CH₂—OC(O)R₁, —CH₂—C(O)—N(R₁)₂, S(O)₂R₁, (O)₂N(R₁)₂, (C₃-C₈)cycloalkyl,(C₃-C₈)cycloalkyl(C₁-C₄)alkyl, SO₃H, and SO₄H;

R₁ is selected from: hydrogen, NH₂, NR₂R₃, OH, —CH₂OH, and —CH₂CH₂OH;

R2 and R3 are independently selected from: hydrogen, (C₁-C₄)alkyl,(C₁-C₄)alkoxy, and (C₁-C₄)cycloalkyl; and

halo is chlorine, bromine, iodine, or fluorine.

For example, compounds of formula VI can be probenecid, as shown below:

Probenecid may be known by the IUPAC name p-[Dipropylsulfamoyl]benzoicacid and has the structure shown above.

In certain embodiments, probenecid and/or an analogue thereof arenonionic, are in the form of a free base, a free acid, or apharmaceutically acceptable salt. By way of example, a pharmaceuticallyacceptable salt includes a hydrochloride salt and salts derived fromacid including, but not limited to, hydrobromic acid, phosphoric acid,acetic acid, fumaric acid, maleic acid, salicylic acid, citric acid,oxalic acid, lactic acid, malic acid, methanesulphonic acid andp-toluene sulphonic acid, a salt of itself. In one embodiment, the saltis a hydrochloride salt. In other embodiments, one or more polymorph,one or more isomer, and/or one or more solvate of probenecid and/or ananalogue thereof may be used.

Panx1

In some aspects, the pannexin modulators can include or exclude pannexinpeptide sequences. The pannexin peptide sequences can comprise 8-40consecutive amino acids, an extracellular domain, an intracellulardomain, a carboxy terminus part, or an amino terminus part, of thepolypeptides Panx1, Panx2 or Panx3. Pannexin modulators may comprise, insome embodiments, a portion of an extracellular loop of Panx 1, 2 or 3.In some embodiments, for example, the pannexin modulator may comprise,for example, the Panx1 mimetic blocking peptide ¹⁰Panx1 (WRQAAFVDSY (SEQID NO: 21)). In some embodiments the pannexin modulators that areoligonucleotides or polynucleotides may have at least about 80%, 85%,90%, 95%, 97%, 98%, or 99% homology to an 8 to 80 nucleotide portion ofSEQ ID NO: 117 (Panx1 polynucleotide), (Panx1 polynucleotide RefSeq IDNM_015368.3), SEQ ID NO:118 (Panx2 polynucleotide), (Panx2polynucleotide RefSeq ID NM_052839.3 for variant 1), SEQ ID NO:119(RefSeq ID NM_001160300.1 for Panx2 polynucleotide variant 2), SEQ IDNO: 120 (RefSeq ID NR_027691.1 for Panx2 polynucleotide variant 3), orSEQ ID NO: 121 (Panx3 polynucleotide) (Panx3 polynucleotide RefSeq IDNM_052959.2). In some aspects, the pannexin modulators can include orexclude pannexin peptide sequences. The pannexin peptide sequences cancomprise 8-40 consecutive amino acids, an extracellular domain, anintracellular domain, a carboxy terminus part, or an amino terminuspart, of the polypeptides SEQ ID NO: 122 (Panx1 peptide), SEQ ID NO: 123(Panx2 peptide), or SEQ ID NO: 124 (Panx3 peptide), or variants thereof.The sequences of Panx1, 2, and 3 polypeptides are shown below.

(Panx1) SEQ ID NO: 122MAIAQLATEYVFSDFLLKEPTEPKFKGLRLELAVDKMVTCIAVGLPLLLISLAFAQEISIGTQISCFSPSSFSWRQAAFVDSYCWAAVQQKNSLQSESGNLPLWLHKFFPYILLLFAILLYLPPLFWRFAAAPHICSDLKFIMEELDKVYNRAIKAAKSARDLDMRDGACSVPGVTENLQSLWEVSESHFKYPIVEQYLKTKKNSNNLIIKYISCRLLTLIIILLACIYLGYYFSLSSLSDEFVCSIKSGILRNDSTVPDQFQCKLIAVGIFQLLSVINLVVYVLLAPVVVYTLFVPFRQKTDVLKVYEILPTFDVLHFKSEGYNDLSLYNLFLEENISEVKSYKCLKVLENIKSSGQGIDPMLLLTNLGMIKMDVVDGKTPMSAEMREEQGNQTAELQGMNIDSETKANNGEKNARQRLLDSSC (Panx2) SEQ ID NO: 123MHHLLEQSADMATALLAGEKLRELILPGAQDDKAGALAALLLQLKLELPFDRVVTIGTVLVPILLVTLVFTKNFAEEPIYCYTPHNFTRDQALYARGYCWTELRDALPGVDASLWPSLFEHKFLPYALLAFAAIIVIYVPALGWEFLASTRLTSELNFLLQEIDNCYHRAAEGRAPKIEKQIQSKGPGITEREKREIIENAEKEKSPEQNLFEKYLERRGRSNFLAKLYLARHVLILLLSAVPISYLCTYYATQKQNEFTCALGASPDGAAGAGPAVRVSCKLPSVQLQRIIAGVDIVLLCVMNLIILVNLIHLFIFRKSNFIFDKLHKVGIKTRRQWRRSQFCDINILAMFCNENRDHIKSLNRLDFITNESDLMYDNVVRQLLAALAQSNHDATPTVRDSGVQTVDPSANPAEPDGAAEPPVVKRPRKKMKWIPTSNPLPQPFKEPLAIMRVENSKAEKPKPARRKTATDTLIAPLLDRSAHHYKGGGGDPGPGPAPAPAPPPAPDKKHARHFSLDVHPYILGTKKAKAEAVPAALPASRSQEGGFLSQAEDCGLGLAPAPIKDAPLPEKEIPYPTEPARAGLPSGGPFHVRSPPAAPAVAPLTPASLGKAEPLTILSRNATHPLLHINTLYEAREEEDGGPRLPQDVGDLIAIPAPQQILIATFDEPRTVVSTVEF (Panx3) SEQ ID NO: 124MSLAHTAAEYMLSDALLPDRRGPRLKGLRLELPLDRIVKFVAVGSPLLLMSLAFAQEFSSGSPISCFSPSNFSIRQAAYVDSSCWDSLLHHKQDGPGQDKMKSLWPHKALPYSLLALALLMYLPVLLWQYAAVPALSSDLLFIISELDKSYNRSIRLVQHMLKIRQKSSDPYVFWNELEKARKERYFEFPLLERYLACKQRSHSLVATYLLRNSLLLIFTSATYLYLGHFHLDVFFQEEFSCSIKTGLLSDETHVPNLITCRLTSLSIFQIVSLSSVAIYTILVPVIIYNLTRLCRWDKRLLSVYEMLPAFDLLSRKMLGCPINDLNVILLFLRANISELISFSWLSVLCVLKDTTTQKHNIDTVVDFMTLLAGLEPSKPKHLTNSACDEHP

The sequences of the mRNA encoding the pannexins are shown below.

(Panx 1 mRNA)  SEQ ID NO: 117 1gggcggcgcg gaggggcagg gccagaggga agcgctttgt tccgcgcgtg gttcccgcgc  61ctgggggtgc gcgggagagg cgcgaatccg agtgccgcgc gcggcccggg gacttgcacg  121ggcgtgcggg gtggaaccgc aggaagcgga gctctcgggt tcccgccccg ccccgccccg  181ccggcggcgg aggcagcgag cgcgagagcc cagcggagtc gctgggagcc tgaggcaccg  241agacacaaag gcaggcggga tgcgggagca ggcaaaggga aagcgaaagc cgcgcgcccg  301gccggtgact gggtgaaggc gccgcgcagc tttcccgacg ccggctgtac ccggacctcc  361tggtcgagcc tggcgcgccg cagccatggc catcgctcaa ctggccacgg agtacgtgtt  421ctcggatttc ttgctgaagg agcccacgga gcccaagttc aaggggctgc gactggagct  481ggctgtggac aagatggtca cgtgcattgc ggtggggctg cccctgctgc tcatctcgct  541ggccttcgcg caggagatct cgattggtac acagataagc tgtttctctc caagttcttt  601ctcctggcgt caggctgcct ttgtggattc atattgctgg gcggctgttc agcagaagaa  661ctcactgcag agcgagtctg gaaacctccc actgtggctg cataagtttt tcccctacat  721cctgctgctc tttgcgatcc tcctgtacct gcccccgctg ttctggcgtt tcgcagctgc  781tcctcatatt tgctcagact tgaagtttat catggaagaa cttgacaaag tttacaaccg  841tgcaattaag gctgcaaaga gtgcgcgtga ccttgacatg agagatggag cctgctcagt  901tccaggtgtt accgagaact tagggcaaag tttgtgggag gtatctgaaa gccacttcaa  961gtacccaatt gtggagcagt acttgaagac aaagaaaaat tctaataatt taatcatcaa  1021gtacattagc tgccgcctgc tgacactcat cattatactg ttagcgtgta tctacctggg  1081ctattacttc agcctctcct cactctcaga cgagtttgtg tgcagcatca aatcagggat  1141cctgagaaac gacagcaccg tgcccgatca gtttcagtgc aaactcattg ccgtgggcat  1201cttccagttg ctcagtgtca ttaaccttgt ggtttatgtc ctgctggctc ccgtggttgt  1261ctacacgctg tttgttccat tccgacagaa gacagatgtt ctcaaagtgt acgaaatcct  1321ccccactttt gatgttctgc atttcaaatc tgaagggtac aacgatttga gcctctacaa  1381tctcttcttg gaggaaaata taagtgaggt caagtcatac aagtgtctta aggtactgga  1441gaatattaag agcagtggtc aggggatcga cccaatgcta ctcctgacaa accttggcat  1501gatcaagatg gatgttgttg atggcaaaac tcccatgtct gcagagatga gagaggagca  1561ggggaaccag acggcagagc tccaaggtat gaacatagac agtgaaacta aagcaaataa  1621tggagagaag aatgcccgac agagacttct ggattcttct tgctgatgat ttttttcctt  1681gagctgtaaa tctgtgactt ctgcgacatg ggatttaatt tggctaaagc acccctgttg  1741gtttcacagc tggtttgcaa taaatggttc ttggtggaga tactgagcat gtcttattga  1801gtccctaatg gaaatggtga tcaacaaaag gttatggaag aatggtttat gaacttccca  1861taggaagcac ctgagagata gtaaactgca gcaagtaact atgtgtaagt cctcatcaaa  1921tgaaaagcag aaagacaaga acaattagtc aagagcagta gccctgtcag agcctcggag  1981caataccttt ctgtacccgt ggtgagacaa gacccagagc tactggaaaa caagcacttt  2041ggaagatttg ttttgttttc atggaataat aatatgtcag ggtataattt aacgtgagtt  2101tcttatgtgc ccttaaagac tgttagacaa gaaaagcatt cactggctaa taatccatag  2161gtcgacctat gtcctaagtt aggtgtaagg tccgatgcct tggcccacac tcgagctctc  2221tttacattgt tagttgtcaa ccttggctga tggaaatccc gtaaccacta tttgttgcac  2281tgtgccttga agggcagcag gcccaagtgc tgctctgact gaaaactgag ttaacaagat  2341gaaatctaaa ggatattcac agtgacttca attcaggaag aatgcttcca aaagagccca  2401gtggggaaat ctgacatcac agaagacatt aattcagtca ctttcaaaga gtttgtctac  2461aggcggtttc tctgttatca aaggcatttg aaataggatt ttacttaaac aataatggaa  2521cacaggagta tttaaagtga agaacacttt gcctgaatgt gatcagggca cataagtgac  2581attggcatgc ttcatatggc gtgcttggag ccagaaaaac ttagcggttt attttgttta  2641tatttaagca cagctttaaa aaattcatta tcgtttattc agtgtccgaa ttgaggccat  2701ttgggaagaa aattctagca ctggtggaga attatagaat aaagattata aatggttgga  2761taagacaaaa aaaaaaaaaa aa  (Panx2 mRNA, transcript variant 1) SEQ ID NO: 118 1atgcaccacc tcctggagca gtcggcggac atggcgaccg cgctgctggc gggagagaag  61ctgcgggagc tgatcctgcc gggcgcgcag gacgacaagg cgggcgcgct ggccgcgctg  121cttctgcagc tgaagctgga gctgccgttc gaccgggtgg tcaccatcgg caccgtgctg  181gtgcccatcc tgctggtcac cctggtcttc accaagaact tcgcagagga acccatttac  241tgttacaccc cgcacaactt cacgcgcgac caggcgctgt acgcccgcgg ctactgctgg  301acggagctgc gggacgcgct gcccggcgtg gacgccagcc tgtggccgtc gctgtttgag  361cacaagttcc tgccctacgc gctgctggcc ttcgccgcca tcatgtacgt gcccgcgctg  421ggctgggagt tcctggcctc cacgcgcctc acctccgagc tcaacttcct gctgcaggag  481atcgacaact gttaccaccg ggcggccgag ggccgcgcgc ccaagatcga gaagcagatc  541cagtccaagg gcccgggcat cacggagcgc gagaagcgcg agatcatcga gaacgcggag  601aaggagaaga gcccggagca gaacctgttc gagaagtacc tggagcgccg cggccgcagc  661aacttcctgg ccaagctgta cctggcgcgg cacgtgctga tcctgctgct gagcgccgtg  721cccatctcct acctgtgcac ctactacgcc acgcagaagc agaacgagtt cacctgcgcg  781ctgggcgcgt ccccggacgg ggcggcaggt gcggggcccg cggtgcgcgt gagctgcaag  841ctcccgtccg tgcaactgca gcgcatcatc gcgggcgtgg acatcgtgct gctgtgcgtc  901atgaacctca tcatcctcgt caacctcatc cacctcttca tcttccgcaa gagcaacttc  961atcttcgaca agctgcacaa ggtgggcatc aagacgcgcc ggcagtggcg ccgctcgcag  1021ttctgcgaca tcaacatcct ggccatgttc tgcaacgaga accgcgacca catcaagtcg  1081ctcaaccggc tggacttcat caccaacgag agcgacctca tgtacgacaa cgtggtccgg  1141cagctgctgg cggcgctggc gcagtccaac cacgacgcca cccccacggt gcgcgactcg  1201ggggtgcaga ccgtggaccc cagcgccaac cccgccgagc ccgacggcgc cgccgagccg  1261cccgtggtca agcggccgcg caagaagatg aagtggatcc ccaccagcaa cccgcttccg  1321cagcccttca aggagccgct ggccatcatg cgcgtggaga acagcaaggc ggagaagccg  1381aagcccgcgc gcaggaagac ggccacggac acgctgatcg cgccgctgct ggaccgctcc  1441gcccaccact acaagggcgg agggggcgac ccgggccccg gccccgcccc tgcccccgcc  1501ccgccgcccg cccctgacaa gaagcacgcg cgccacttct ccctggacgt gcacccctac  1561atcctcggca ccaagaaggc caaggccgag gcggtgcccg ccgccctgcc cgcctcccgg  1621agccaggagg ggggcttcct gtcccaggcg gaggactgtg ggctaggcct ggccccggcg  1681cccatcaaag atgctccgct ccccgagaag gaaatcccgt accccacaga gccagcccgg  1741gcagggcttc cctcgggggg cccgttccac gtccgctcac ctcccgccgc ccctgctgtg  1801gcccctctga caccagccag cctgggcaag gcggagcccc tcaccatcct gagccgaaac  1861gccacacacc cgctgctgca catcaacacg ctgtacgagg cccgggagga ggaggacggg  1921ggcccccgcc tgccgcagga cgtgggggac ctcatcgcca tccctgcccc acagcagatc  1981ctcatcgcca ccttcgacga gccgagaacg gtcgtgagta ctgtggagtt ttgagggatg  2041gcaccgtcca ggccgccgag agcccctctg cctgtgtcgt gtggcctggc cagcctcccg  2101gtggacacca gccctgcgtg gacgtggcct gtgcttcgcc cgcactgcgc gcatccccaa  2161cctctgtccg catgcctggg gccttcgccc ccacgtgctc gacaggggaa cccgcccgga  2221cggcatcgcc aggcactggc tggggtgggg aaaggtggcc cagtggagcc ggtggccagg  2281aaggctgaag cccgcttccc atgctcctgc atcaggtgcc cagccgtggg tgggggccct  2341gaggtgaaga gtttattttt ttagtccgtt tcgtcctggc cccgggctgt ggcgagacag  2401cccaactccc ccagcccagc tcccccagcc cagagccagg gaagaggaag gtggggccag  2461tcccaccagt ggggtggcca cgcccatggg gtcacatgct caggggtcac cccctgcagg  2521gacctgatgc cctcgggtgg gagggaccga ggtccaccct cgggtcaaag gtcaacgtgc  2581actttctcct tgtcgcctga cagacatttt attttactaa gactgctgta ccgaacaagc  2641atatttatca tcaggagaca ggatgggttt aaagcaggat ggtgtgtgtg tgaacgggca  2701tgagcagagg tgagcgtgag cgagcgggtg tgtatgtacg agtgtgcacg tgtgtgcgtg  2761tgcacagagg gtgtggtgcc agcttgagtg ggagtgtgtg agtgtgagca ggcgggcgag  2821tgcgtgagtg cacgccagcg cgtggcccat gtatgaggag tgaaggggcc caacgcaata  2881accacgtccc ccacccgggc cccccgccgc ggctgaggcc acatggcttc ctgtgggagc  2941cccggccggc acccggctgg tcccacccca aatacctcag ccatggagac catgtcatgc  3001agaattaaca aggtagcacc gagcatatca ataaatatta ttctgataat caaaaaaaaa  3061aaaaaaaaa  (Panx2 mRNA, transcript variant 2)  SEQ ID NO: 119 1atgcaccacc tcctggagca gtcggcggac atggcgaccg cgctgctggc gggagagaag  61ctgcgggagc tgatcctgcc gggcgcgcag gacgacaagg cgggcgcgct ggccgcgctg  121cttctgcagc tgaagctgga gctgccgttc gaccgggtgg tcaccatcgg caccgtgctg  181gtgcccatcc tgctggtcac cctggtcttc accaagaact tcgcagagga acccatttac  241tgttacaccc cgcacaactt cacgcgcgac caggcgctgt acgcccgcgg ctactgctgg  301acggagctgc gggacgcgct gcccggcgtg gacgccagcc tgtggccgtc gctgtttgag  361cacaagttcc tgccctacgc gctgctggcc ttcgccgcca tcatgtacgt gcccgcgctg  421ggctgggagt tcctggcctc cacgcgcctc acctccgagc tcaacttcct gctgcaggag  481atcgacaact gttaccaccg ggcggccgag ggccgcgcgc ccaagatcga gaagcagatc  541cagtccaagg gcccgggcat cacggagcgc gagaagcgcg agatcatcga gaacgcggag  601aaggagaaga gcccggagca gaacctgttc gagaagtacc tggagcgccg cggccgcagc  661aacttcctgg ccaagctgta cctggcgcgg cacgtgctga tcctgctgct gagcgccgtg  721cccatctcct acctgtgcac ctactacgcc acgcagaagc agaacgagtt cacctgcgcg  781ctgggcgcgt ccccggacgg ggcggcaggt gcggggcccg cggtgcgcgt gagctgcaag  841ctcccgtccg tgcaactgca gcgcatcatc gcgggcgtgg acatcgtgct gctgtgcgtc  901atgaacctca tcatcctcgt caacctcatc cacctcttca tcttccgcaa gagcaacttc  961atcttcgaca agctgcacaa ggtgggcatc aagacgcgcc ggcagtggcg ccgctcgcag  1021ttctgcgaca tcaacatcct ggccatgttc tgcaacgaga accgcgacca catcaagtcg  1081ctcaaccggc tggacttcat caccaacgag agcgacctca tgtacgacaa cgtggtccgg  1141cagctgctgg cggcgctggc gcagtccaac cacgacgcca cccccacggt gcgcgactcg  1201ggggtgcaga ccgtggaccc cagcgccaac cccgccgagc ccgacggcgc cgccgagccg  1261cccgtggtca agcggccgcg caagaagatg aagtggatcc ccaccagcaa cccgcttccg  1321cagcccttca aggagccgct ggccatcatg cgcgtggaga acagcaaggc ggagaagccg  1381aagcccgcgc gcaggaagac ggccacggac acgctgatcg cgccgctgct ggaccgctcc  1441gcccaccact acaagggcgg agggggcgac ccgggccccg gccccgcccc tgcccccgcc  1501ccgccgcccg cccctgacaa gaagcacgcg cgccacttct ccctggacgt gcacccctac  1561atcctcggca ccaagaaggc caaggccgag gcggtgcccg ccgccctgcc cgcctcccgg  1621agccaggagg ggggcttcct gtcccaggcg gaggactgtg ggctaggcct ggccccggcg  1681cccatcaaag atgctccgct ccccgagaag gaaatcccgt accccacaga gccagcccgg  1741gcagggcttc cctcgggggg cccgttccac gtccgctcac ctcccgccgc ccctgctgtg  1801gcccctctga caccagccag cctgggcaag gcggagcccc tcaccatcct gagccgaaac  1861gccacacacc cgctgctgca catcaacacg ctatcctcat cgccaccttc gacgagccga  1921gaacggtcgt gagtactgtg gagttttgag ggatggcacc gtccaggccg ccgagagccc  1981ctctgcctgt gtcgtgtggc ctggccagcc tcccggtgga caccagccct gcgtggacgt  2041ggcctgtgct tcgcccgcac tgcgcgcatc cccaacctct gtccgcatgc ctggggcctt  2101cgcccccacg tgctcgacag gggaacccgc ccggacggca tcgccaggca ctggctgggg  2161tggggaaagg tggcccagtg gagccggtgg ccaggaaggc tgaagcccgc ttcccatgct  2221cctgcatcag gtgcccagcc gtgggtgggg gccctgaggt gaagagttta tttttttagt  2281ccgtttcgtc ctggccccgg gctgtggcga gacagcccaa ctcccccagc ccagctcccc  2341cagcccagag ccagggaaga ggaaggtggg gccagtccca ccagtggggt ggccacgccc  2401atggggtcac atgctcaggg gtcaccccct gcagggacct gatgccctcg ggtgggaggg  2461accgaggtcc accctcgggt caaaggtcaa cgtgcacttt ctccttgtcg cctgacagac  2521attttatttt actaagactg ctgtaccgaa caagcatatt tatcatcagg agacaggatg  2581ggtttaaagc aggatggtgt gtgtgtgaac gggcatgagc agaggtgagc gtgagcgagc  2641gggtgtgtat gtacgagtgt gcacgtgtgt gcgtgtgcac agagggtgtg gtgccagctt  2701gagtgggagt gtgtgagtgt gagcaggcgg gcgagtgcgt gagtgcacgc cagcgcgtgg  2761cccatgtatg aggagtgaag gggcccaacg caataaccac gtcccccacc cgggcccccc  2821gccgcggctg aggccacatg gcttcctgtg ggagccccgg ccggcacccg gctggtccca  2881ccccaaatac ctcagccatg gagaccatgt catgcagaat taacaaggta gcaccgagca  2941tatcaataaa tattattctg ataatcaaaa aaaaaaaaaa aaaa (Panx2 mRNA, transcript variant 3)  SEQ ID NO: 120 1atgcaccacc tcctggagca gtcggcggac atggcgaccg cgctgctggc gggagagaag  61ctgcgggagc tgatcctgcc gggcgcgcag gacgacaagg cgggcgcgct ggccgcgctg  121cttctgcagc tgaagctgga gctgccgttc gaccgggtgg tcaccatcgg caccgtgctg  181gtgcccatcc tgctggtcac cctggtcttc accaagaact tcgcagggtg gacgctcttc  241tctggctcct gggattggct gtgaggacaa aacataaagg aacccattta ctgttacacc  301ccgcacaact tcacgcgcga ccaggcgctg tacgcccgcg gctactgctg gacggagctg  361cgggacgcgc tgcccggcgt ggacgccagc ctgtggccgt cgctgtttga gcacaagttc  421ctgccctacg cgctgctggc cttcgccgcc atcatgtacg tgcccgcgct gggctgggag  481ttcctggcct ccacgcgcct cacctccgag ctcaacttcc tgctgcagga gatcgacaac  541tgttaccacc gggcggccga gggccgcgcg cccaagatcg agaagcagat ccagtccaag  601ggcccgggca tcacggagcg cgagaagcgc gagatcatcg agaacgcgga gaaggagaag  661agcccggagc agaacctgtt cgagaagtac ctggagcgcc gcggccgcag caacttcctg  721gccaagctgt acctggcgcg gcacgtgctg atcctgctgc tgagcgccgt gcccatctcc  781tacctgtgca cctactacgc cacgcagaag cagaacgagt tcacctgcgc gctgggcgcg  841tccccggacg gggcggcagg tgcggggccc gcggtgcgcg tgagctgcaa gctcccgtcc  901gtgcaactgc agcgcatcat cgcgggcgtg gacatcgtgc tgctgtgcgt catgaacctc  961atcatcctcg tcaacctcat ccacctcttc atcttccgca agagcaactt catcttcgac  1021aagctgcaca aggtgggcat caagacgcgc cggcagtggc gccgctcgca gttctgcgac  1081atcaacatcc tggccatgtt ctgcaacgag aaccgcgacc acatcaagtc gctcaaccgg  1141ctggacttca tcaccaacga gagcgacctc atgtacgaca acgtggtccg gcagctgctg  1201gcggcgctgg cgcagtccaa ccacgacgcc acccccacgg tgcgcgactc gggggtgcag  1261accgtggacc ccagcgccaa ccccgccgag cccgacggcg ccgccgagcc gcccgtggtc  1321aagcggccgc gcaagaagat gaagtggatc cccaccagca acccgcttcc gcagcccttc  1381aaggagccgc tggccatcat gcgcgtggag aacagcaagg cggagaagcc gaagcccgcg  1441cgcaggaaga cggccacgga cacgctgatc gcgccgctgc tggaccgctc cgcccaccac  1501tacaagggcg gagggggcga cccgggcccc ggccccgccc ctgcccccgc cccgccgccc  1561gcccctgaca agaagcacgc gcgccacttc tccctggacg tgcaccccta catcctcggc  1621accaagaagg ccaaggccga ggcggtgccc gccgccctgc ccgcctcccg gagccaggag  1681gggggcttcc tgtcccaggc ggaggactgt gggctaggcc tggccccggc gcccatcaaa  1741gatgctccgc tccccgagaa ggaaatcccg taccccacag agccagcccg ggcagggctt  1801ccctcggggg gcccgttcca cgtccgctca cctcccgccg cccctgctgt ggcccctctg  1861acaccagcca gcctgggcaa ggcggagccc ctcaccatcc tgagccgaaa cgccacacac  1921ccgctgctgc acatcaacac gctatcctca tcgccacctt cgacgagccg agaacggtcg  1981tgagtactgt ggagttttga gggatggcac cgtccaggcc gccgagagcc cctctgcctg  2041tgtcgtgtgg cctggccagc ctcccggtgg acaccagccc tgcgtggacg tggcctgtgc  2101ttcgcccgca ctgcgcgcat ccccaacctc tgtccgcatg cctggggcct tcgcccccac  2161gtgctcgaca ggggaacccg cccggacggc atcgccaggc actggctggg gtggggaaag  2221gtggcccagt ggagccggtg gccaggaagg ctgaagcccg cttcccatgc tcctgcatca  2281ggtgcccagc cgtgggtggg ggccctgagg tgaagagttt atttttttag tccgtttcgt  2341cctggccccg ggctgtggcg agacagccca actcccccag cccagctccc ccagcccaga  2401gccagggaag aggaaggtgg ggccagtccc accagtgggg tggccacgcc catggggtca  2461catgctcagg ggtcaccccc tgcagggacc tgatgccctc gggtgggagg gaccgaggtc  2521caccctcggg tcaaaggtca acgtgcactt tctccttgtc gcctgacaga cattttattt  2581tactaagact gctgtaccga acaagcatat ttatcatcag gagacaggat gggtttaaag  2641caggatggtg tgtgtgtgaa cgggcatgag cagaggtgag cgtgagcgag cgggtgtgta  2701tgtacgagtg tgcacgtgtg tgcgtgtgca cagagggtgt ggtgccagct tgagtgggag  2761tgtgtgagtg tgagcaggcg ggcgagtgcg tgagtgcacg ccagcgcgtg gcccatgtat  2821gaggagtgaa ggggcccaac gcaataacca cgtcccccac ccgggccccc cgccgcggct  2881gaggccacat ggcttcctgt gggagccccg gccggcaccc ggctggtccc accccaaata  2941cctcagccat ggagaccatg tcatgcagaa ttaacaaggt agcaccgagc atatcaataa  3001atattattct gataatcaaa aaaaaaaaaa aaaaa  (Panx3 mRNA)  SEQ ID NO: 121 1atgtcacttg cacacacagc tgcagagtac atgctctcag atgccctgct gcctgaccgc  61aggggacccc gcctcaaagg actgcgtctg gaactgcccc tggaccggat agtcaagttc  121gtagctgtgg gctccccctt gttgctgatg tccctggcat tcgcccagga gttctcctct  181gggtctccga tcagctgctt ctctcccagt aacttcagca tccggcaggc agcctacgtg  241gacagctcct gctgggactc actgcttcac cataagcagg acgggcctgg ccaggacaaa  301atgaaatctc tctggcccca caaggccctc ccctactccc tgctggccct ggccttgctc  361atgtacctgc cggtgctgct gtggcagtat gcagctgtgc cagccctcag ctccgatctg  421ctgttcatca tcagcgaact ggacaaatct tataatcgct ccatccgcct cgtgcagcac  481atgctgaaga tccggcagaa gagttccgac ccctatgtgt tctggaatga gctggagaag  541gctcggaaag aacgatactt tgaattccct ttgctagagc ggtacctggc atgtaagcag  601cgttcacatt cgctagtggc tacctacctc ctgaggaact ccctcttgct catcttcacc  661tccgccactt acctatacct tggtcatttc catctggatg tcttcttcca ggaagaattc  721agctgctcca tcaagacagg gctgctaagt gatgagaccc atgtccccaa tctgatcaca  781tgcaggctga catcactgtc cattttccag attgttagcc tctccagtgt agcaatatac  841accatattgg ttccagtgat aatatacaac ctcacacggc tatgtcggtg ggacaaacga  901cttttatctg tctatgagat gctcccagct tttgatctcc tcagcagaaa gatgctagga  961tgtcccatca atgacctcaa tgtgatcctt cttttcctcc gagctaacat ctctgagctc  1021atctctttta gctggctgag tgtcttatgt gtgttgaagg atacaaccac ccagaagcac  1081aatattgaca cagtagttga ttttatgact ttattggctg gcttagaacc ctcaaaaccc  1141aaacacctca ccaactcggc atgtgatgaa cacccatagt taagaaacca tggagcaaga  1201aagcttgtgg aaagtctctc tccttcctca taagacatgc acactaatac acatacacac  1261caaaaaatta cacattttaa aactgctaag cttggattta actgaatcat atatctttta  1321tcatgttatc ctaaaagtga gaagacataa ccaagacatg gaaataaatg tgaaagctgg  1381agccgaagag tcaaagagct aaaaaattaa gtctagaaca ttctatgagg atagtataaa  1441taaaaagaaa tacagtctag acatgctgca aggaaagaag attctaaagt ccgtttatgg  1501aggcaattcc atatcctttc ttgaacgcac attcagctta ccccagagag caagtgaggc  1561aatctggcaa aagattaata aagatgtaaa cccctggaaa aaaaaaaaa 

Connexins and pannexins are the hemichannel-forming proteins invertebrates. In one aspect this invention relates to pharmaceuticalcompositions, articles of manufacture, and methods for treating ocularand other disorders, for example, glaucoma, DME, AMD, DR, ocularfibrosis, ocular hypoxia, retinal perfusion impairment, and/orneuropathic ocular disorders by administering a therapeuticallyeffective amount of at least one connexin modulator to the eye of saidsubject. In some aspects the neuropathic ocular disorder may be, forexample, loss of retinal ganglion cells and/or glaucomatous opticneuropathy. In some aspects, administering a therapeutically effectiveamount of at least one connexin modulator is effective for stopping,preventing, or treating loss of retinal ganglion cells are furtheruseful for increasing the levels of neurotrophins in the glaucomatousoptic nerve and decreasing vitreal glutamate concentrations.

The major risk factor for most glaucomas is increased intraocularpressure, i.e. ocular hypertension. Intraocular pressure is a functionof production of liquid aqueous humor by the ciliary processes of theeye, and its drainage through the trabecular meshwork. Aqueous humorflows from the ciliary processes into the posterior chamber, boundedposteriorly by the lens and the zonules of Zinn, and anteriorly by theiris. It then flows through the pupil of the iris into the anteriorchamber, bounded posteriorly by the iris and anteriorly by the cornea.From here, the trabecular meshwork drains aqueous humor via Schlemm'scanal into scleral plexuses and general blood circulation. However, inother glaucoma patients, normal tension glaucoma may be present.

In open/wide-angle glaucoma, flow is reduced through the trabecularmeshwork, due to the degeneration and obstruction of the trabecularmeshwork, whose original function is to absorb the aqueous humor. Lossof aqueous humor absorption leads to increased resistance and thus achronic, painless buildup of pressure in the eye. In close/narrow-angle,the iridocorneal angle is completely closed because of forwarddisplacement of the final roll and root of the iris against the cornea,resulting in the inability of the aqueous fluid to flow from theposterior to the anterior chamber and then out of the trabecularnetwork. This accumulation of aqueous humor causes an acute increase ofpressure and pain.

The inconsistent relationship of glaucoma with ocular hypertension hasprovoked studies on anatomic structure, eye development, nervecompression trauma, optic nerve blood flow, excitatory neurotransmitter,trophic factor, retinal ganglion cell/axon degeneration, glial supportcell, immune system, aging mechanisms of neuron loss, and severing ofthe nerve fibers at the scleral edge. As featured herein, in someembodiments, the gap junction, connexin and/or pannexin modulatorsfeatured herein are useful in treating or preventing ocular disorders asdisclosed herein, including, for example, conditions such ashypertensive and normatensive glaucoma and for other uses featuredherein, treating or preventing DME or ocular fibrosis, or for treatingAMD from its earliest stages through its later stages.

Age-related macular degeneration (ARMD or AMD) is another leading causeof irreversible visual impairment, and the leading cause of visualimpairment in people over the age of 60 years in developed nations. AMDmost commonly affects the macula, an area of the retina that subservesfine and detailed vision (but may not be confined to the macular). Inthe United States one in seven of the population over 50 showneovascular AMD and/or geographic atrophy with 1.8 million citizenshaving AMD (and expected to rise to 3 million by 2020). Friedman, et al.(2004) Prevalence of age-related macular degeneration in the UnitedStates, Arch Ophthalmol 122: 564-572. The incidence of AMD increasesdramatically with age, however, and more than 15% of white women olderthan 80 years having neovascular AMD and/or geographic atrophy.Worldwide AMD costs are estimated at $345B per annum with $255B of thatin direct health care costs. AMD was included in the 2010 eye diseasepriority list by the World Health Organization as a disease withoutsatisfactory treatment options.

The etiology of AMD is most likely to be multifactorial, involving acomplex interaction of metabolic, genetic and environmental factors. Aconventional understanding of AMD is that it affects four functionallyinterrelated layers in the eye: the photoreceptors, the RPE, Bruch'smembrane, and the choriocapillaris. Shelley, et al. (2009) Conedegeneration in aging and age-related macular degeneration, ArchOphthalmol 127: 483-492. Research has primarily focused on thedegeneration of the RPE which leads to irreversible damage to thephotoreceptors. Nowak (2006) Age-related macular degeneration (AMD):pathogenesis and therapy, Pharmacol Rep 58: 353-363. Along with thisdegeneration, deposits (Drusen) can accumulate within the RPE andBruch's membrane. These deposits can be associated with abnormal bloodvessel ingrowth, which in turn can lead to leakage, bleeding andultimately scar formation at the macula. See de Jong P T (2006)Age-related macular degeneration, N Engl J Med 355: 1474-1485; Finger,et al. (1999) Ophthalmic plaque radiotherapy for age-related maculardegeneration associated with subretinal neovascularization, Am JOphthalmol 127: 170-177.

In one aspect this invention relates to pharmaceutical compositions,articles of manufacture, and methods for treating ocular disorders, forexample, glaucoma, DME, AMD, DR ocular fibrosis, and/or ocular hypoxiaand/or retinal perfusion impairment, and/or neuropathic ocular disordersintraocular pressure-associated neuropathies, for example, byadministering a therapeutically effective amount of at least oneconnexin modulator and/or at least one pannexin modulator to the eye ofsaid subject. In one aspect, this invention relates, for example topharmaceutical compositions and methods for treating glaucoma. Themethods herein provide for treatment of intraocular pressure-associatedoptic neuropathy such as glaucoma, in an amount sufficient to reduceintraocular pressure. In some aspects, the connexin modulators areuseful in treating trauma associated with elevated intraocular pressure.In some aspects, the connexin modulator is a connexin 43 modulator. Insome aspects, the compositions and methods of this invention are usefulin reducing the intraocular pressure to normal levels, e.g., below 21 mmHg, for example to below 21, 20 or 19 mm Hg, for example to levelsbetween about 8 and about 21 mm Hg.

The compositions, articles of manufacture and methods described hereinare useful, in one aspect, to treat glaucoma without toxic side effects.In some aspects, the glaucoma may be open-angle glaucoma orangle-closure glaucoma. In some aspects, administering a therapeuticallyeffective amount of at least one pannexin modulator and/or at least oneconnexin modulator, for example, a connexin 43 channel modulator, to theeye tissue in need thereof increases flow through the trabecularmeshwork. Also useful are gap junction modulators, pannexin channelmodulators, and hemichannel modulators. In some aspects, thecompositions of this invention are useful as adjuvants to improvetrabeculectomy success rates.

The front of the eye is filled with aqueous humor, a clear fluid thatprovides nourishment to the structures in the anterior portion of theeye. This fluid is produced constantly by the ciliary body, whichsurrounds the lens of the eye. The aqueous humor flows through the pupiland out of the eye through the trabecular meshwork channels located atthe junction where the cornea attaches to the iris, which is referred toas the drainage angle of the eye. In some aspects of this invention, oneor more gap junction or connexin modulators, or panenxin modulators, forexample, a connexin 43 modulator, is administered at or near thetrabecular meshwork or the ciliary body. Also useful are gap junctionmodulators, pannexin channel modulators, and hemichannel modulators.

Also featured in one aspect of this invention are compositions, articlesof manufacture, and methods for treating retinal perfusion impairment,retinal ischemic diseases or optic ischemic diseases in a subject,comprising administering a therapeutically effective amount of a gapjunction and/or connexin modulator, and/or a pannexin or pannexinchannel modulator, effective to reduce inflammation in the inner retina.In some aspects, the retinal ischemic disease is retinal arteryocclusion, or central retinal vein occlusion. In some aspects, the opticischemic disease is, for example, anterior ischemic optic neuropathy. Insome aspects the connexin modulator is a modulator of Cx43, Cx26, Cx30,Cx31.1, Cx36, Cx37, Cx40, Cx45, Cx50, Cx57 or any other connexin in theeye or blood vessels. In some aspects, the connexin modulator is a Cx43modulator, for example a Cx43 hemichannel modulator. In some aspects,the modulator can include or exclude any of the foregoing.

This invention also features compositions, articles of manufacture, andmethods for treating ocular disorders, for example, glaucoma, AMD,ocular fibrosis, and/or ocular hypoxia and/or retinal perfusionimpairment, and/or neuropathic ocular disorders intraocularpressure-associated neuropathies, by, for example, reducing impairmentof choridal perfusion and/or choroidal inflammation, or choroidaloverperfusion in a subject, comprising administering an amount of a gapjunction and/or connexin modulator and/or a pannexin or pannexin channelmodulator to the choroid of the subject, effective to reduce impairmentof choridal perfusion and/or choroidal inflammation, or choroidaloverperfusion and/or inflammation in the inner retina. In some aspectsof this invention, administering a therapeutically effective amount of agap junction and/or connexin modulator to the choroid of the subjecteffective to reduce impairment of choridal perfusion and/or choroidalinflammation, or choroidal overperfusion, also reduces choriocapillarisendothelial cell loss and/or choriocapillaris dropout. In some aspects,reducing impairment of choridal perfusion and/or choroidal inflammation,or choroidal overperfusion also reduces retinal pigmental epitheliumdegeneration and/or Drusen development, or otherwise ameliorates, stops,slows, and/or reverses the progression of macular degeneration ormacular dystrophy, which can be dry macular degeneration or wet maculardegeneration. The connexin modulator or pannexin modulator for reducingimpairment of choridal perfusion and/or choroidal inflammation, orchoroidal overperfusion may also be administered with an oculartreatment agent. In some embodiments, the modulator for reducingimpairment of choridal perfusion and/or choroidal inflammation, orchoroidal overperfusion is a Cx43 modulator, hemichannel modulator,pannexin modulator or pannexin channel modulator.

In some aspects of the methods of this invention, the gap junctionmodulator and/or connexin modulator, or pannexin or pannexin channelmodulator may be administered to the eye by intraocular injection, orintravitreal injection. The gap junction modulator and/or connexinmodulator, pannexin or pannexin channel modulator may be administeredonce, or more than once. Other methods of administering modulators arefeatured herein.

In some embodiments the connexin modulators of this invention includeconnexin oligonucleotides or polynucleotides, such as connexinanti-sense oligonucleotides or polynucleotides, or connexin peptides orpeptidomimetics, such as connexin peptides or peptidomimetics. Connexinpeptides or peptidomimetics comprise, for example, any of the peptidesdescribed herein, including peptides comprising a portion of anextracellular domain of a connexin, and peptides comprising a portion ofa carboxy-terminal portion of a connexin or gap junction closingcompounds, and hemichannel closing compounds useful in the methods ofthis invention, such as healing any of the ocular disorders describedherein.

In some embodiments the connexin protein modulating agents of thisinvention include connexin 43 oligonucleotides or polynucleotides, suchas connexin 43 anti-sense oligonucleotides or polynucleotides, oranti-connexin 43 peptides or peptidomimetics, for example, any of thepeptides described herein, including peptides comprising a portion of anextracellular domain of a connexin, and peptides comprising a portion ofa carboxy-terminal portion of a connexin or gap junction closingcompounds, and hemichannel closing compounds useful in the methods ofthis invention, such as healing any of the neuropathic ocular disordersdescribed herein.

In some embodiments the pannexin modulators of this invention includepannexin oligonucleotides or polynucleotides, such as pannexinanti-sense oligonucleotides or polynucleotides, or anti-pannexinpeptides or peptidomimetics useful in the methods of this invention,such as healing any of the ocular disorders, such as glaucoma, AMD,ocular fibrosis, DME, ocular hypoxia and/or neuropathic ocular disordersdescribed herein.

In some embodiments “promoiety” refers to a species acting as aprotecting group which masks a functional group within an active agent,thereby converting the active agent into a pro-drug. The active agentmay be any of the modulators or ocular therapeutics disclosed herein.Typically, the promoiety will be attached to the drug via bond(s) thatare cleaved by enzymatic or non-enzymatic means in vivo, therebyconverting the pro-drug into its active form. In some embodiments thepromoiety may also be an active agent. In some embodiments the promoietymay be bound to a gap junction modulator, a connexin modulator, or apannexin modulator. In some embodiments the promoiety may be bound toany of the polynucleotides, peptides or peptidomimetics, small moleculeantagonists and/or ocular treatments disclosed herein. In someembodiments the promoeity may be bound to a compound of Formula I. Insome embodiments the pro-drug may be a compound of Formula II.

In some embodiments the promoiety may be any peptidomimetic or peptideantagonist of this disclosure. In some embodiments, the promoeity is asingle amino acid which is optionally protected on its functionalgroups. In some embodiments, the promoeity is a targeting species. Insome aspects, the promoeity is a substrate for an influx or effluxtransporters on the cell membrane, for example those described inGaudana, R. et al. The AAPS Journal, 12:3, 348-360 (2012). The promoeitycan be, for example, chemically-linked biotin. The promoeity can be, forexample, chemically-linked D-serine.

In some embodiments the pannexin modulators of this invention includepannexin oligonucleotides or polynucleotides, such as pannexinanti-sense oligonucleotides or polynucleotides, or anti-pannexinpeptides or peptidomimetics useful in the methods of this invention,such as healing any of the ocular hypoxia or neuropathic oculardisorders described herein.

Definitions

A “small molecule” is defined herein to have a molecular weight belowabout 600 daltons, and is generally an organic compound. A smallmolecule can be an active agent of a prodrug.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual, tissue or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment include,but are not limited to, preventing occurrence or recurrence of adisease, disorder or condition, alleviation of signs or symptoms,diminishment of any direct or indirect pathological consequences of thedisease, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome embodiments, compounds, methods and compositions of the inventioncan be used to delay development of a disease, disorder or condition, orto slow the progression of a disease, disorder or condition. The termdoes not necessarily imply that a subject is treated until totalrecovery. Accordingly, “treatment” includes reducing, alleviating orameliorating the symptoms or severity of a particular disease, disorderor condition or preventing or otherwise reducing the risk of developinga particular disease, disorder or condition. It may also includemaintaining or promoting a complete or partial state of remission of acondition.

The term “treating ocular disorders” or the like, including diseases andconditions, may refer to preventing, slowing, reducing, decreasing,stopping and/or reversing the ocular disorder, disease or condition,such as, for example, neuronal loss and/or neuropathy, vessel leakand/or hemorrhage, neovascularization, inflammation and/or edema in theeye.

The term “preventing” means preventing in whole or in part, orameliorating or controlling.

As used herein, “effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic or prophylactic result. For example, and not by way oflimitation, an “effective amount” can refer to an amount of a compoundor composition, disclosed herein, that is able to treat the signs and/orsymptoms of a disease, disorder or condition.

As used herein, “therapeutically effective amount” of asubstance/molecule of the invention, agonist or antagonist may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the substance/molecule, agonist orantagonist to elicit a desired response in the individual. Atherapeutically effective amount is preferably also one in which anytoxic or detrimental effects of the substance/molecule, agonist orantagonist may be outweighed by the therapeutically beneficial effects.

As used herein, “prophylactically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve adesired prophylactic result. Typically but not necessarily, since aprophylactic dose is used in subjects prior to or at an earlier stage ofa disease, disorder or condition, the prophylactically effective amountwill be less than the therapeutically effective amount.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which does not containadditional components that are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier,” as used herein, refers to aningredient in a pharmaceutical formulation, other than an activeingredient, which can be safely administered to a subject. Apharmaceutically acceptable carrier includes, but is not limited to, abuffer, excipient, stabilizer, or preservative.

As used herein, the term “subject” or the like, including “individual,”and “patient”, all of which may be used interchangeably herein, refersto any mammal, including humans, domestic and farm animals, and zoo,wild animal park, sports, or pet animals, such as dogs, horses, cats,sheep, pigs, cows, etc. The preferred mammal herein is a human,including adults, children, and the elderly. Preferred sports animalsare horses and dogs. Preferred pet animals are dogs and cats. Thesubject may be, for example, an aquatic park animal, such as a dolphin,whale, seal or walrus. In certain embodiments, the subject, individualor patient is a human.

The invention is described herein with reference to the use of a gapjunction channel modulator, including modulators such as peptide 5and/or an analogue thereof, compounds of formula I, for exampletonabersat, and/or an analogue or pro-drug of any of the foregoingcompounds, and/or the use of a pannexin modulator, e.g., probenecidand/or a synthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1, or an analogue of either thereof. It should be appreciated that oneor more salt, polymorph, solvate and/or isomer of a gap junction channelmodulator such as peptide 5 and/or an analogue thereof, compounds offormula I, for example tonabersat, and/or an analogue or pro-drug of anyof the foregoing compounds, and/or a pannexin modulator, e.g.,probenecid and/or a synthetic mimetic peptide blocker of pannexin 1,e.g., ¹⁰Panx1, or an analogue of either, may also be used in theinvention. Accordingly, references to “tonabersat and/or an analoguethereof,” “peptide 5 and/or an analogue thereof,” “probenecid and/or ananalogue thereof,” or “¹⁰Panx 1, or an analogue of either thereof,”should be taken to include reference to any one or more salts, solvates,polymorphs, and/or isomers of an of the foregoing compounds.

Methods, uses and compositions of the invention may comprise the use ofa combination of compounds of formula I, for example tonabersat, and/oran analogue of any of the foregoing compounds and one or more analoguesthereof, peptide 5 and one or more analogues thereof, probenecid and oneor more analogues thereof, or ¹⁰Panx1 and one or more analogues thereof,or a combination of one or more of these analogues. Accordingly,reference herein to “compounds of formula I, for example tonabersat,and/or an analogue,” “peptide 5 and/or an analogue thereof,” “compoundsof formula VI, for example probenecid, and/or an analogue thereof,” or“¹⁰Panx 1, or an analogue of either thereof,” should be taken to includereference to such combinations.

As used herein, the term “hemichannel” is a part of a gap junction (twohemichannels or connexons connect across an intercellular space betweenadjacent cells to form a gap junction) and is comprised of a number ofconnexin proteins, typically homo- or hetero-meric hexamers of connexinproteins that form the pore for a gap junction between the cytoplasm oftwo adjacent cells. The hemichannel is supplied by a cell on one side ofthe junction, with two hemichannels from opposing cells normally comingtogether to form the complete intercellular gap junction channel.However, in some cells, and in cells under some circumstances, thehemichannel itself is active as a conduit between the cytoplasm and theextracellular space allowing the transference of ions and smallmolecules.

Compounds of formula I, for example tonabersat, and/or an analogue orpro-drug of any of the foregoing compounds, can modulate the functionand/or activity of hemichannels, preferably those comprising any type ofconnexin protein. Accordingly, reference to “hemichannel” should betaken broadly to include a hemichannel comprising any one or more of anumber of different connexin proteins, unless the context requiresotherwise. However, by way of example, a hemichannel may comprise one ormore of connexin23, 25, 26, 30, 30.2, 30.3, 31, 31.1, 31.9, 32, 36, 37,40, 40.1, 43, 45, 46, 47, 50, 59, and 62. In one embodiment, ahemichannel consists of one of the aforementioned connexins. In oneembodiment, a hemichannel comprises one or more of connexin26, 30, 32,36, 37, 40, 45 and 47. In one embodiment, a hemichannel consists of oneof connexin26, 30, 32, 36, 37, 40, 45 or 47. In one particularembodiment, a hemichannel comprises one or more of connexin30 andconnexin43. In one particular embodiment, a hemichannel consists of oneof connexin30 or connexin43. In one particular embodiment, a hemichannelconsists of one of connexin45 or connexin46 or connexin50 In oneembodiment, the hemichannel comprises one or more connexin excludingconnexin26.

As used herein, the term “gap junction channel” comprises twohemichannels which connect across an intercellular space betweenadjacent cells and allow certain molecules to flow between those cells.

Hemichannels and gap junction channels may be present in cells of anytype. Accordingly, reference to a “hemichannel” or a “gap junctionchannel” should be taken to include reference to a hemichannel or gapjunction channel present in any cell type, unless the context requiresotherwise. In one embodiment of the invention, the hemichannel or gapjunction channel is present in a cell outside of the central nervoussystem. In one embodiment, the hemichannel or gap junction channelcomprises one or more connexin excluding connexin 26 and is present in acell outside the central nervous system. In one embodiment of theinvention, the hemichannel or gap junction channel is present in a cellin the eye. In one embodiment of the invention, the hemichannel or gapjunction channel is present in a cell in the front of the eye, i.e., theanterior segment. In one embodiment of the invention, the hemichannel orgap junction channel is present in a cell in the back of the eye, i.e.,the posterior segment. In one embodiment of the invention, thehemichannel or gap junction channel is present in a cell in the uvea. Inone embodiment of the invention, the hemichannel or gap junction channelis present in a cell in the optic nerve. In one embodiment of theinvention, the hemichannel or gap junction channel is present in a cellin the microvasculature of the eye. In one embodiment of the invention,the hemichannel or gap junction channel is present in a cell in thechoroid. In one embodiment of the invention, the hemichannel or gapjunction channel is present in a cell in the choriocapillaris.

As used herein, “modulation of a hemichannel and/or a gap junctionchannel” is the modulation of one or more functions and/or activities ofa hemichannel and/or gap junction channel. Such functions and activitiesmay include, for example, docking of hemichannels on adjacent cells andopening to form a gap junction channel. They may also includeintercellular communication between cells, and the flow of moleculesbetween cells through a gap junction channel. Such functions andactivities may include, for example, the flow of molecules from theextracellular space or environment through a hemichannel into a cell,and/or the flow of molecules through a hemichannel from theintracellular space or environment of a cell into the extracellularspace or environment.

Modulation of the function of a hemichannel and/or a gap junctionchannel may occur by any means. However, by way of example only,modulation may occur by one or more of: preventing, blocking, inhibitingor decreasing the formation of a gap junction through hemichanneldocking; inducing or promoting closure of a hemichannel; preventing,blocking, inhibiting or decreasing hemichannel opening; inducing orpromoting loss of coupling between hemichannels; triggering, inducing orpromoting cellular internalization of a hemichannel and/or gap junction.Use of the words such as “blocking”, “inhibiting”, “preventing”,“decreasing” and “antagonizing”, and the like, should not be taken toimply complete blocking, inhibition, prevention, or antagonism, althoughthis may be preferred, and should be taken to include partial blocking,inhibition, prevention or antagonism to at least reduce the function oractivity of a hemichannel and/or gap junction channel. Similarly,“inducing” or “promoting” should not be taken to imply complete loss ofcoupling or complete internalization of a hemichannel and/or gapjunction (or group of hemichannels and/or gap junctions), and should betaken to include partial loss of coupling or partial internalization toat least reduce the function or activity of a hemichannel and/or gapjunction channel.

As used herein, the term “gap junction channel modulator” is a compoundthat prevents, inhibits, and/or reduces the function or activity of agap junction channel or the function or activity of a gap junctionhemichannel, together or separately, including, for example, prevention,inhibition and/or reduction in expression, activity and/or the formationof hemichannels and/or gap junctions, including the expression of aconnexin protein, its trafficking and/or assembly. Prevention,inhibition and/or reduction of function or activity may be direct orindirect (for example, but not limited to, directly blocking a channel,inducing a conformational change, or modifying a connexinphosphorylation state). The gap junction channel blocker may be of anychemical nature. However, by way of example, the agent may be a nucleicacid (including antisense molecules, RNAi molecules, morpholinos, andother nucleic acids as described herein), a peptide, a small molecule, achemical element, hormone, antibody, antibody fragment or a metabolite.In certain embodiments, it is a compound that targets one or morecomponent of a gap junction, including connexins, hemichannels (alsoknown as connexons), to inhibit or block its activity, expression,trafficking and/or assembly. “Inhibits” or “blocks” should not be takento imply that the activity, expression, trafficking and/or assembly of aconnexin, hemichannel or gap junction is completely inhibited orblocked, although this may be preferred, but should be taken to includeany reduction in the activity, expression, trafficking and/or assemblyof a connexin, hemichannel or gap junction.

As used herein, the term “disorder where modulation of a gap junctionhemichannel and/or gap junction channel may be of benefit” includes anydisease, disorder or condition in which gap junction channel and/orhemichannel function or activity may be implicated in the onset,progression, or persistence of the disease, disorder or condition. Inone embodiment, the disease, disorder or condition is one in which thefunction or activity of a gap junction channel and/or hemichannelcomprising one or more of connexins23, 25, 26, 30, 30.2, 30.3, 31, 31.1,31.9, 32, 36, 37, 40, 40.1, 43, 45, 46, 47, 50, 59, and 62 isimplicated. In one embodiment, the disease, disorder or condition is onein which the function or activity of a gap junction channel and/orhemichannel consisting of one of the aforementioned connexins isimplicated. In one embodiment, the disease, disorder or condition is onein which the function or activity of a gap junction channel and/orhemichannel comprising one or more of connexin26, 30, 32, 36, 37, 40, 45and 47 is implicated. In one embodiment, the disease, disorder orcondition is one in which the function or activity of a gap junctionchannel and/or hemichannel consisting of one of connexin26, 30, 32, 36,37, 40, 45 or 47 is implicated. In one particular embodiment, thedisease, disorder or condition is one in which the function or activityof a gap junction channel and/or hemichannel comprising one or more ofconnexin 30 and connexin 43 is implicated. In one particular embodiment,the disease, disorder or condition is one in which the function oractivity of a gap junction channel and/or hemichannel consisting of oneof connexin30 or connexin43 or connexin45 or connexin46 or connexin50 isimplicated. In one embodiment, the disease, disorder or condition is onein which the function or activity of a gap junction channel and/orhemichannel comprising one or more connexin excluding connexin26 isimplicated. In one embodiment, the disease, disorder or condition is onein which the function or activity of a gap junction channel and/orhemichannel present in a cell outside of the central nervous system isimplicated. In one embodiment, the disease, disorder or condition is onein which the function or activity of a gap junction channel and/orhemichannel comprising one or more connexins excluding connexin26 and ispresent in a cell outside the central nervous system is implicated. Inone embodiment of the invention, the disease, disorder or condition isone in which the function or activity of the hemichannel or gap junctionchannel to be modulated is present in a cell in the eye. In oneembodiment of the invention, the disease, disorder or condition is onein which the function or activity of the hemichannel or gap junctionchannel to be modulated is present in a cell in the front of the eye,i.e., the anterior segment. In one embodiment of the invention, thedisease, disorder or condition is one in which the function or activityof the hemichannel or gap junction channel to be modulated is present ina cell in the back of the eye, i.e., the posterior segment. In oneembodiment of the invention, the disease, disorder or condition is onein which the function or activity of the hemichannel or gap junctionchannel to be modulated is present in a cell in the uvea. In oneembodiment of the invention, the disease, disorder or condition is onein which the function or activity of the hemichannel or gap junctionchannel to be modulated is present in a cell in the optic nerve. In oneembodiment of the invention, the disease, disorder or condition is onein which the function or activity of the hemichannel or gap junctionchannel to be modulated is present in a cell in the choroid. In oneembodiment of the invention, the disease, disorder or condition is onein which the function or activity of the hemichannel or gap junctionchannel to be modulated is present in a cell in the choriocapillaris. Inone embodiment of the invention, the disease, disorder or condition isone in which the function or activity of the hemichannel or gap junctionchannel to be modulated is present in a cell in the microvasculature ofthe eye. In one embodiment, the composition can include or exclude anygap junction channel and/or hemichannel modulator of the foregoing.

As used herein, the term “disorder where modulation of a pannexinchannel may be of benefit” should be taken to include any disorder inwhich pannexin or pannexin channel function or activity may beimplicated in the onset, progression, or persistence of a disease,disorder or condition. In one embodiment, the disease, disorder orcondition is one in which the function or activity of a pannexin channelcomprises one or more of the three isoforms of pannexins. In oneembodiment, the disease, disorder or condition is one in which thefunction or activity of a pannexin channel to be modulated comprisespannexin 1. In one embodiment, the disease, disorder or condition is onein which the function or activity of a pannexin channel to be modulatedis in or on the eye, including the surface of the eye, the retina, andvessels associated with or comprising the eye.

“Chronic wounds”, “wounds that do not heal at the/an expected rate”, and“dehiscent wounds” and like terms and phrases, as used herein, have themeaning as provided in US2011/0300130, which is herein incorporated byreference and includes a number of examples of the types of wounds towhich the present invention may be applied. However, by way of example,such wounds may include diabetic ulcers (including for example, diabeticfoot ulcers), venous ulcers, venous stasis ulcers, pressure ulcers,decubitus ulcers, vasculitic ulcers, arterial ulcers, infectious ulcers,pressure ulcers, burn ulcers, traumatic or trauma-induced ulcers,inflammatory ulcers, ulcerations associated with pyoderma gangrenosum,ocular ulcers, including persistent epithelial defects, mixed ulcers.

“Fibrosis” and “fibrotic diseases, disorders or conditions” or liketerms and phrases, as used herein, have the meaning as provided inUS2011/0092449, which is herein incorporated by reference and includes anumber of examples of fibrosis, diseases, disorders or conditions towhich the present invention may be applied. However, by way of example,such fibrosis, fibrotic diseases, disorders or conditions may includeliver fibrosis, cardiac fibrosis, lung fibrosis (including for example,silicosis, asbestosis, idiopathic pulmonary fibrosis), oral fibrosis(including for example, oral submucous fibrosis), retroperitonealfibrosis, deltoid fibrosis, endomyocardial fibrosis, kidney fibrosis(including for example, diabetic nephropathy), glomerulosclerosis, acutefibrosis. In one embodiment, the liver fibrosis has arisen from chronicliver injury or is associated with haemochromatosis, Wilson's disease,alcoholism, schistosomiasis, viral hepatitis, bile duct obstruction,exposure to toxins, and/or metabolic disorders. In one embodiment, thecardiac fibrosis is endocardial fibrosis or endomyocardial fibrosis. Inone embodiment, the acute fibrosis is associated an accidental injury,an infection, and/or radiation and/or chemotherapy treatments. In oneembodiment, the fibrosis may occur in a subject that has a disease,disorder or condition selected from the group comprising scleroderma,pancreatitis, inflammatory bowel disease, Crohn's disease, nodularfacilitis and/or eosinophilic facilitis. In one embodiment, thescleroderma may be morphea, generalized morphea, or linear scleroderma.

“Abnormal or excessive scarring” and the like, as used herein, have themeaning as provided in US2011/0130710, which is herein incorporated byreference and includes a number of examples of abnormal or excessivescarring to which the present invention may be applied. However, by wayof example, abnormal or excessive scarring may include scars present inor on the skin, scars in or on the eye, a keloid scar, a hypertrophicscar, an atrophic scar, a widespread scar.

“Vascular disorders” and like terms or phrases, as used herein, have themeaning as provided in EP2510939, which is herein incorporated byreference and includes a number of examples of vascular disorders towhich the present invention may be applied. However, by way of example,vascular disorders may include atherosclerosis, microvascular disorders,macrovascular disorders, thrombosis, vascular injuries resulting fromtrauma, vascular damage, diabetic retinopathy, organ ischemia,endothelial cell disruption, vascular diseases of the extremities.

An “orthopedic disease or disorder” and like phrases, as used herein,have the meaning as provided in EP2238250, which is herein incorporatedby reference and includes a number of examples orthopedic diseases ordisorder to which the present invention may be applied. However, by wayof example, the orthopedic disease or disorder includes one which ischaracterized in whole or in part by abnormal tissue formation insideand/or around a joint, one which is associated with altered or abnormaljoint mobility or joint architecture which may be associated with orcaused by a variety of injuries and conditions such as, for example,metabolic disorders, ischemia, trauma, injury to joint, capsule, bone,cartilage, tendon, ligament or muscle, fractures, subluxation,dislocation, crush injuries, prolonged immobilization (e.g.,immobilization of a joint in a cast or splint), and/or paralysis.

The phrase an “orthopedic surgery or procedure” and like phrases, asused herein, will be readily understood by persons skilled in the art.However, by way of example, it should be taken to include any surgery orprocedure outlined in EP2242844. “Improving” the recovery from such aprocedure should also be taken broadly and may include, for example,reducing pain, and improving mobility and/or recovery time.

“Post-orthopedic surgical joint contracture” and like phrases, as usedherein, have the meaning as provided in EP2242844, which is hereinincorporated by reference.

“Adhesions” and like phrases, as used herein, have the meaning asprovided in EP2252690, which is herein incorporated by reference.However, by way of example, it may include surgical adhesions andadhesions which form in any tissue including epithelia, connectivetissue, muscle, and tissue.

“Tissue damage”, “tissue damage associated with an ophthalmic procedure”and like phrases and terms, as used herein, have the meaning as providedin US2012/0093768, which is herein incorporated by reference andincludes a number of examples of tissue damage to which the presentinvention may be applied. However, by way of example, tissue damage mayinclude enhancing tissue repair processes and/or ameliorating tissuedamage.

An “inflammatory disorder”, “inflammatory disease” and like phrases, asused herein, have the meaning as provided in provided in WO2013/148736,which is herein incorporated by reference and includes a number ofexamples of disorders to which the present invention may be applied. Inone embodiment, hemichannel and pannexin modulators are used, alone ortogether, to inhibit activation of one or more inflammasomes. In oneembodiment, modulators including hemichannel and pannexin or pannexinchannel modulators are used, alone or together, to inhibit activation ofan inflammatory cascade by an inflammasome. In one embodiment,modulators including gap junction, hemichannel and pannexin and/orpannexin channel modulators are used, alone or together, to treat asubject for a disease, disorder or condition characterized, at least inpart, by the activation of one or more inflammasomes and/or by theactivation of an inflammatory cascade by an inflammasome. In oneembodiment, gap junction, hemichannel, pannexin channel, connexin andpannexin modulators are used, alone or together, to modulate activity ofthe NLRP3 inflammasome

The inflammasome is a multiprotein complex comprising caspase 1, PYCARD,NALP, and optionally caspase 5 (also known as caspase 11 or ICH-3). Theexact composition of an inflammasome depends on the activator thatinitiates inflammasome assembly. For example, dsRNA will trigger oneinflammasome composition whereas asbestos will assemble a differentvariant. Inflammasomes promote the maturation of the inflammatorycytokines Interleukin 1β (IL-1β) and Interleukin 18 (IL-18).Inflammasomes are responsible for activation of inflammatory processes,and has been shown to induce cell pyroptosis, a process of programmedcell death distinct from apoptosis. The NLRP3 inflammasome blockadeinhibits VEGF-A-induced age-related macular degeneration.

The terms “modulating agent,” “modulator” and “modulation” of a gapjunction channel, a hemichannel, a pannexin or pannexin channel, orconnexin function or activity, as used herein in its various forms,refers to inhibition in whole or in part of the expression, action oractivity of a connexin or a connexin hemichannel or connexin gapjunction or a pannexin or pannexin channel, in whole or in part, and mayfunction as anti-connexin agents, including as gap junction modulationagents, and as anti-pannexin agents, including as pannexin channelmodulation agents. In some aspects the gap junction and/or connexinmodulator may be a modulator of a connexin present in blood vessels, forexample, a connexin 43 modulator and/or a connexin 45 modulator. Thus,as used herein, the term “connexin modulator” refers generally toconnexin modulators, but also specifically to connexin 43 modulators andconnexin 45 modulators (and Cx43 and Cx45 hemichannel modulators), andmodulators of other blood vessel connexins and hemichannels, unlessotherwise provided. In some aspects, the connexin modulator is aconnexin43 modulator, e.g., a connexin43 hemichannel modulator thatblocks hemichannel opening. In some aspects the gap junction modulatoralso includes modulators of other connexins found in the eye, such asCx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx50 and Cx57, and theirhemichannels and gap junctions. In some aspects the pannexin and/orpannexin channel modulator may be a modulator of pannexin 1,particularly a modulator of pannexin 1 channel opening. In some aspects,the modulating agent can include or exclude any of the foregoing.

In some embodiments, “ocular disorders” include or exclude glaucomaincluding hypertensive glaucoma and normatensive glaucoma, clinicalgeographic atrophy; AMD including dry AMD and exudative AMD,abnormalities or impairment, chronic macular ischemia, fibrosis of theeye, idiopathic polypoidal choroidopathy (IPC); diabetic maculopathy,diabetic retinopathy; hypertensive retinopathy, inflammatory CNV;central serous chorioretinopathy (CSR); macular telangiectasia; patterndystrophy; subretinal/subPRD neovascularization; serous detachment ofthe neurosensory retina; RPE detachment; hemorrhages (subretinal pigmentepithelial, subretinal, intraretinal or pre-retinal, includingbreakthrough bleeding into the vitreous); piretinal, intraretinal,subretinal or sub-pigment epithelial scar/glial tissue or fibrin-likedeposits; retinal fibrosis, retinal angiomatous proliferations andretinochoroidal anatastamosis; choroidal neovascularization (CNV);cystic maculopathy; retinal thickening; non-exudative AMD; and retinalscarring, uveitis, including posterior uveitis, scleritis, episcleritisviral retinitis, including cytomegalovirus (CMV) retinitis, retinopathyof prematurity, retinal hypoxia, diffuse choroidal sclerosis, sclerosisof the choriocapillaris, dry eye, ocular and corneal persistentepithelial defects, diabetic macular edema (DME), neuropathic oculardisorders, trauma induced lowering of intraocular pressure, epithelialbasement membrane dystrophy, and/or other ocular disorders, includingthose noted elsewhere herein.

As used herein, “ocular neuropathy” or “neuropathic ocular disorder” isany ocular disorder, disease, or condition that would benefit from anagent that treats ocular neuronal loss, such as stopping or reducingloss of RGCs or damage or loss of other ocular neurons or structures, orrestoring lost RGCs or other ocular structures or neurons. Also includedare disorders in connection with neuropathic, ischemic, perfusionimpairment, inflammatory or microvascular pathology in the eye, whichmay result from choroidal or retinal perfusion impairment, ocularhypertension, inflammation of the choroid or inner retina, or otherinternal tissues of the eye, or retinal vein or artery occlusion. Insome embodiments, chroroidal or retinal perfusion impairment may resultin ocular hypoxia. Also included are any ocular diseases, disorders andconditions characterized by elevated expression of a connexin present ineye blood vessels or neurons. Also included are ocular diseases,disorders and conditions characterized by unwanted pannexin activity.Also included are diseases, disorders and conditions characterized byunwanted ZO-1 protein or ZO-1 protein activity or that would benefitfrom reduced ZO-1 protein or ZO-1 protein activity. Also included arediseases, disorders and conditions characterized by unwanted lower Rac1or Rac1 activity or that would benefit from increased Rac1 or Rac1activity. Also included are diseases, disorders and conditionscharacterized by unwanted reduced RhoA GTPase or RhoA GTPase activity orthat would benefit from reduced RhoA GTPase or RhoA GTPase activity.

“Glaucomatous ocular neuropathy” refers to any “ocular neuropathy”associated with glaucoma, for example, neuropathic damage to retinalganglion cells (RPGs) and glaucomatous optic neuropathy associated withglaucoma. In some embodiments, glaucomatous optic neuropathy may or maynot be associated with elevated intraocular pressure or choroidal orretinal perfusion impairment. In some embodiments, methods of treatingneovascular glaucoma include treatment by administering to a subject inneed, a modulator, including a gap junction modulator, a pannexinmodulator, a pannexin channel modulator and/or a connexin modulator.

The term “intraocular pressure-associated neuropathy” refers toglautomatous optic neuropathy associated with ocular hypertension, i.e.,elevated intraocular pressure.

The term “ocular hypoxia” refers to any eye condition or disorderresulting from hypoxia, including from choroidal or retinal perfusionimpairment, hypertension, or interruption of blood flow and/or oxygenflow to the eye, including, for example, ischemia and/or blood vesselleakage and/or blood vessel breakdown in the eye. Conditions that resultfrom ocular hypoxia include, for example, glaucoma, glaucomatous ocularneuropathy intraocular pressure associated neuropathy, ocularneuropathy, clinical geographic atrophy, DME, choriocapillaris dropoutand/or breakdown of capillaries in the choroid or other conditionsdescribed herein and below.

Clinical Geographic Atrophy (GA)

The presentation of GA is usually insidious and often detected duringroutine fundus examination. When GA is bilateral and involves the foveaof both eyes, patients may complain of deterioration of central vision.A common mode of presentation is difficulty with reading initially withthe smallest sizes of print and then later with larger print and orwords. The confirmation of the diagnosis of GA is by clinicalexamination using a high definition fundus lens for stereobiomicroscopy. This will reveal the characteristic area or areas ofpallor with sharply defined and scalloped edges. When the area of GA islarger than 500 microns, large choroidal vessels are clearly visiblewithin the area of pallor.

Usually areas of drusen and focal hyperpigmentation are visible in theretina adjacent to the patch of GA. Several imaging modalities may beuseful, in particular fundus autofluorescence, in the evaluation of GA.Fundus autofluorescence along with spectral domain OCT has made iteasier to diagnose GA, as these imaging modalities can reveal areas ofGA that may not be clinically visible on biomicroscopy.

Geographic atrophy is the advanced (late) form of dry AMD. Here, atrophyrefers to the degeneration of the deepest cells of the retina. Usuallydefined as any sharply delineated round or oval area ofhypopigmentation, or apparent absence of the retinal pigment epithelium(RPE), in which choroidal vessels are more visible than in surroundingareas. The most common sequence of events leading to GA is theprogression of a large drusen to hyperpigmentation, followed byregression of the drusen, hypopigmentation and ultimately RPE celldeath, with development of an atrophic area of retina and underlyingchoriocapillaris, sometimes preceded by the appearance of refractiledeposits.

In some embodiments, the modulator, e.g. a gap junction and/or connexinmodulator, or pannexin or pannexin channel modulator preventsinflammation of the choroid vessels, maintains the retinal pigmentepithelium cell layer, and/or prevents atrophy in the area of the retinaand underlying choriocapillaris.

Exudative Age-Related Macular Degeneration (AMD)

The commonest symptoms typical of onset of exudative AMD are centralvisual blurring and distortion. Most patients will complain thatstraight lines appear crooked or wavy. Sometimes patients do not noticevisual symptoms when the first eye is affected. When exudative AMDoccurs in the second eye, patients suddenly become unable to read,drive, and see fine detail such as facial expressions and features. Thissymptom described by many with AMD of a central dark patch in the visualfield noticed at night, which clears within a few minutes as they adapt.This symptom can also be present in patients with AMD who do notnecessarily develop exudative AMD.

Examination of the macula usually reveals an exudative macular lesionalong with other features of early AMD such as drusen and pigmentaryirregularities. Sometimes these latter features are not visible onceexudative AMD has supervened. However the fellow eye, if free ofadvanced disease, will often exhibit some or all of these early clinicalsigns and their presence is helpful in supporting the diagnosis that theneovascular lesion is due to AMD. Following slit lamp biomicroscopy thepresence or absence of the following signs should be noted: (1)subretinal or sub-RPE neovascularisation which may be visible as greygreen lesions (occasionally the lesion will have a dark pigmented edgewhich is thought to be due to proliferation of the RPE at the edge ofthe membrane); serous detachment of the neurosensory retina; (3) RPEdetachment; (4) hemorrhages—subretinal pigment epithelial, subretinal,intraretinal or pre-retinal (breakthrough bleeding into the vitreous mayalso occur); (5) hard exudates (lipids) within the macular area relatedto any of the above, and not related to other retinal vascular disease;(6) piretinal, intraretinal, subretinal or sub-pigment epithelialscar/glial tissue or fibrin-like deposits; (7) retinal angiomatousproliferations and retinochoroidal anatastamosis. This disorder may betreated with a modulator, e.g. a gap junction and/or connexin modulator,or pannexin or pannexin channel modulator, as described herein.

Idiopathic Polypoidal Choroidopathy (IPC)

This is an atypical form of neovascular AMD in which highly exudativelesions with steep walled hemorrhagic pigment epithelial detachments areseen most typically adjacent to the optic disc, but can occur anywherewithin the macula and even outside the macula. High speed fluorescein orindocyanine green angiography typically reveals hyperfluorescent dilatedcomplexes of choroidal vessels (branching vascular networks) that leakin the later phases of the angiograms. These dilated complexes look likepolyps or grapes and hence the name. It was originally described inmiddle aged black populations and was more commonly seen in females. IPCis considered part of the spectrum of AMD and a strong association withhypertension and ischemic heart disease has been described. The use ofconfocal high speed imaging devices allows IPC to be diagnosed morefrequently and IPC accounts for more than a third of serosanguinousmaculopathy in older adults in Asian populations and for 8-13% of thatseen in Caucasians. This disorder may also be treated with a modulator,e.g. a gap junction and/or connexin modulator, or pannexin or pannexinchannel modulator, as described herein.

Ocular Fibrosis

Disruption of the highly ordered tissue architecture in the eye causedby vascular leakage, hemorrhage, and concomitant fibrosis can lead tomechanical disruption of the visual axis and/or biologicalmalfunctioning. Ocular fibrosis can also include, for example, fibrosisof the lens, macula or retina, and may include, for example, progressivesubretinal fibrosis and premacular fibrosis (PMF). Macular fibrosisoccurs when a thin sheet of fibrotic scar tissue forms on top of themacula, in response to damage or injury. Damage to the macula may occurdue to eye trauma, retinal tears or detachments, the shrinking of thevitreous, or systemic disease, such as diabetes or hypertension. Macularfibrosis may also be referred to as a macular pucker, epiretinalmembrane, or cellophane maculopathy. Subretinal fibrosis may beassociated with chronic vitreous inflammation resulting in fibroticsubretinal leasions which pregressively enlarge and coalesce, and may beassociated with cystoid macular edema. PMF which affects the macula, onthe front side of the retina, whereas macular degeneration affects theunderside of the retina. The primary symptom of PMF is the gradualdevelopment of distortion in vision in one eye, which may develop overweeks to months. This disorder can be treated with a modulator, e.g. agap junction and/or connexin modulator, or pannexin or pannexin channelmodulator, as described herein.

Uveitis

Uveitis is inflammation of the uvea, the middle layer of the eye. Theuvea consists of the middle, pigmented vascular structures of the eye,and includes the iris, choroid and ciliary body. The choroid issandwiched between the retina and the white of the eye (sclera), andprovides blood flow to the deep layers of the retina. Uveitis mayinclude inflammation of the iris called iritis (anterior uveitis). Thisdisorder can also be treated with a modulator, e.g. a gap junctionand/or connexin modulator, or pannexin or pannexin channel modulator, asdescribed herein.

Diabetic Maculopathy

This is the most common exudative central macular disorder in olderadults. Patients with diabetes frequently exhibit retinalmicroaneurysms, hemorrhages and exudates often in the context of macularedema. The presence of more extensive vascular signs outside the maculararcade along with venous engorgement or beading should alert theclinician to a diagnosis of diabetic maculopathy. The visual function isless markedly reduced in eyes with diabetic maculopathy when compared toeyes with CNV involving the fovea. Blood vessels near to the macula leakfluid or protein onto the macula. Fluorescein angiography is needed toconfirm the absence of choroidal neovascularisation and sub RPEpathology. Sometimes exudative AMD and diabetic maculopathy can coexistas both are common conditions. This disorder can be treated with amodulator, e.g. a gap junction and/or connexin modulator, or pannexin orpannexin channel modulator, as described herein, as can high myopiaassociated with choroidal neovascularization.

High myopia can be associated with choroidal neovascularisation. Theseneovascular complexes are believed to occur as a consequence of thedevelopment of minute cracks in thinned Bruch's membrane allowingchoroidal vessels to access the subretinal space.

Diabetic Macular Edema

Diabetic macular edema is swelling of the retina in subjects withdiabetes due to leaking of fluid from blood vessels within the macula.The macula is the central portion of the retina, a small area rich incones, the specialized nerve endings that detect color and upon whichdaytime vision depends. As macular edema develops, blurring occurs inthe middle or just to the side of the central visual field. Visual lossfrom diabetic macular edema can progress over a period of months andmake it impossible to focus clearly. Macular edema in common indiabetes. The lifetime risk for diabetics to develop macular edema isabout 10%. The condition is closely associated with the degree ofdiabetic retinopathy (retinal disease). Hypertension (high bloodpressure) and fluid retention also increase the hydrostatic pressurewithin capillaries which drives fluid from within the vessels into theretina. A common cause of fluid retention in diabetes is kidney diseasewith loss of protein in the urine (proteinuria). Diabetic macular edemais classified into focal and diffuse types. This is an importantdifference because the two types differ in treatment. Focal macularedema is caused by foci of vascular abnormalities, primarilymicroaneurysms, which tend to leakage fluid whereas diffuse macularedema is caused by dilated retinal capillaries in the retina. Thisdisorder can also be treated with a modulator, e.g. a gap junctionand/or connexin modulator, or pannexin or pannexin channel modulator, asdescribed herein.

Diabetic Retinopathy

Diabetic retinopathy can eventually lead to blindness. It is the ocularmanifestation of a systemic disease and afflicts up to 80% of allpatients who have had diabetes for at least 10 years. In diabeticretinopathy hyperglycemia induces pericyte death and basement membranethickening leading to compromise of vascular walls. This damage altersthe blood-retinal barrier to make retinal blood vessels permeable. Smallblood vessels such as in the eye are especially vulnerable to poor bloodsugar control. Abnormal connexin expression in diabetes is associatedwith complications in several tissues including the skin, kidneys,bladder, perineurium, lens and heart 76. In the retina, hyperglycemia isknown to induce apoptosis leading to vascular drop out and pericyteloss, hallmarks of background diabetic retinopathy. Recent evidenceshows that reduced Cxn43 expression initiates the apoptosis and thebreakdown of vascular homeostasis (Bobbie M W, Roy S, Trudeau K, MungerS J, Simon A M, Roy S, Invest Ophthalmol Vis Sci.; 51(7): 3758-63,2010). The above description, however, refers in the main to theunderlying background of the disease during the initial stage ofnon-proliferative diabetic retinopathy and most patients will not noticeany change in their vision, although some will develop macular edemawhen damaged blood vessels leak fluid and lipids onto the macula. As thedisease progresses, diabetic retinopathy enters a blood vesselproliferation stage. Owing to low oxygen levels in the retina the newvessels are fragile and grow along the retina and into the vitreoushumour. These vessels can bleed, cloud vision, and destroy the retina.Fibrovascular proliferation can cause retinal detachment and vessels canalso grow into the angle of the anterior chamber of the eye, causingneovascular glaucoma. Diabetic retinopathy has prominent features ofchronic, subclinical inflammation (Zhang W, Liu H, Rojas M, Caldwell RW, Caldwell R B, Immunotherapy, 3(5): 609-28, 2011. This disorder can betreated with a modulator, e.g. a gap junction and/or connexin modulator,or pannexin or pannexin channel modulator, as described herein. In someembodiments, the modulator can also treat this inflammatory phase ofdiabetic retinopathy.

Inflammatory CNV

A number of the choroidal inflammatory white dot syndromes (e.g.presumed ocular histoplasmosis, punctate inner choroidopathy, multifocalinner choroidopathy) can be associated with inflammatory choroidalneovascularisation. In some embodiments, modulator as described hereinprevents inflammation of the choroid, thereby preventing inflammatoryCNV.

Central Serous Chorioretinopathy (CSR)

This is characterized by a collection of serous fluid in thesub-neurosensory retina without any evidence of neovascularisation.Chronic CSR can sometimes be confused with AMD, again the history,symptoms and a combination of retinal imaging usually helps distinguishbetween the two. CNV and IPCV can occur as a complication of chronicCSR. It is characterized by leakage of fluid under the retina that has apropensity to accumulate under the central macula. This allows choroidalfluid to leak into the subretinal space. The build-up of fluid occursbecause of small breaks in the retinal pigment epithelium. In someembodiments, the modulators described herein prevent or otherwiseameliorate inflammation of the choroid, and/or prevents or ameliorateschoiroidal capillarisis dropout. Retention of the choroid can preventleakage of choroidal fluid into the subretinal space.

Macular Telangiectasia

Idiopathic macular telangiectasia (MACTEL) also sometimes termedperifoveal or juxtafoveal telangiectasia may be difficult to distinguishfrom nAMD, particularly with the RAP form of n AMD. Two types oftelangiecgtasia have been described: Type 1 MACTEL occurs in middle agepersons; the condition is usually unilateral and exhibits exudativefeatures as the vessels are leaky and intraretinal fluid accumulationoccurs with a cystic maculopathy and surrounding exudates. Type 2 MACTELoccurs in older people and is usually bilateral with evidence ofcrystalline deposits, pigmentary charges, and right angled venulesevident temporal to the fovea and extending to the entire perifovealregion. While leakage is detectable on fluorescein angiography, there isno evidence of increased retinal thickening. Cystic spaces are evidentwithin the retina using OCT and these spaces are thought to reflect theloss of retinal tissue. Occasionally, sub-retinal neovascularizationdevelops and arises from the retinal circulation. Macular telangiectasiadevelops when there are problems with the tiny blood vessels around thefovea, the center of the macula. There are two types of maculartelangiectasia, and each affects the blood vessels differently. Type 2macular telangiectasia: The most common form of macular telangiectasiais Type 2 macular telangiectasia, in which the tiny blood vessels aroundthe fovea leak, become dilated (widen), or both. In some cases, newblood vessels form under the retina and they can also break or leak.Fluid from leaking blood vessels causes the macula to swell or thicken,a condition called macular edema, which affects your central vision.Also, scar tissue can sometimes form over the macula and the fovea,causing loss of detail vision. Type 2 affects both eyes but notnecessarily with the same severity. Type 1 macular telangiectasia: InType 1 macular telangiectasia, the blood vessels become dilated formingtiny aneurysms, causing swelling and damaging macular cells. The diseasealmost always occurs in one eye, which differentiates it from Type 2.These disorders can also be treated with a modulator, e.g. a gapjunction and/or connexin modulator, or pannexin or pannexin channelmodulator, as described herein.

Pattern Dystrophy (PD)

PD affects the macula and can be mistaken for nonexudative AMD. The mostcommon types of PD seen are adult vitelliform macular dystrophy (AVMD)and less commonly butterfly shaped pattern dystrophy. PD is a conditionthat has a genetic basis; although a family history is often notpresent. PD is usually associated with a better visual outcome than AMD,unless complicated by choroidal neovascularisation or atrophic changes.Differentiating AVMD in particular from AMD can be difficult. Symptomsmay be similar particularly if CNV or atrophy complicates PD, but oftenAVMD is identified in an asymptomatic individual at a routinefundoscopic review. Fundus autofluorecence imaging especially whencombined with optical coherence tomography is helpful in distinguishingPD from AMD. Fluorescein angiography can show a typical ‘corona sign’ inAVMD, and the branching lines seen in butterfly shaped PD are associatedwith a hyperfluorescence distributed in the area of the deposits, whichdoes not show leakage throughout the phases of the angiogram.Occasionally, fluorescein angiographic staining of the vitelliformlesion can be mistaken for active leakage from CNV. This disorder can betreated with a modulator, e.g. a gap junction and/or connexin modulator,or pannexin or pannexin channel modulator, as described herein.

Subretinal/subPRD Neovascularization

Subretinal neovascularization is a pathological process consisting ofthe formation of new blood vessels in the choroid. In the wet form ofAMD, abnormal blood vessels grow under the macula and leak fluid andblood. The abnormal vessels, called subretinal neovascularization, mayalso lift up the retina. This disorder can be treated with a modulator,e.g. a gap junction and/or connexin modulator, or pannexin or pannexinchannel modulator, as described herein.

Serous Detachment of the Neurosensory Retina

Retinal detachment occurs when subretinal fluid accumulates between theneurosensory retina and the retinal pigment epithelium. This process canoccur in three ways. One mechanism involves a break in the retinaallowing vitreous to directly enter the subretinal space. This is knownas a rhegmatogenous retinal detachment. A second mechanism involvesproliferative membranes on the surface of the retina or vitreous. Thesemembranes can pull on the neurosensory retina causing a physicalseparation between the neurosensory retina and retinal pigmentepithelium. This is called a traction retinal detachment. The thirdmechanism for retinal detachment is due to accumulation of subretinalfluid due to inflammatory mediators or exudation of fluid from a masslesion. This mechanism is known as a serous or exudative retinaldetachment. Serous detachments are caused by a number of inflammatory,or exudative retinal disease processes such as Sarcoidosis or choroidalneoplasms. In some aspects, a modulator described herein is used tocontrol inflammation in the underlying choriocapillaris, controlsbuildup of subretinal fluid, and/or prevents an accumulation ofsubretinal fluid, which can lead, for example, to serous detachment ofthe neurosensory retina.

RPE Detachment

RPE detachment is a non-specific anatomical alteration that may resultfrom any number of choroidal disorders that disrupt the normal junctionbetween the basement membrane of the RPE and the inner collagenous layerof Bruch's membrane. This disruption permits serous fluid from theunderlying choriocapillaris to gain access into the sub-RPE space.Age-related macular degeneration, choroidal neovascular membranes, highmyopia, angioid streaks, hereditary choroidal degeneration, POHS, andtumors of the choroid have all been identified as precipitatingconditions in the development of RPE detachment. This disorder can betreated with a modulator, e.g. a gap junction and/or connexin modulator,or pannexin or pannexin channel modulator, as described herein.

Hemorrhages—Subretinal Pigment Epithelial, Subretinal, Intraretinal orPre-Retinal, Including Breakthrough Bleeding into the Vitreous

Retinal hemorrhage is a disorder of the eye in which bleeding occursinto the retensitive tissue on the back wall of the eye. A retinalhemorrhage can be caused by hypertension, retinal vein occlusion (ablockage of a retinal vein), or diabetes mellitus (which causes smallfragile blood vessels to form, which are easily damaged). Retinalhemorrhages, especially mild ones not associated with chronic disease,will normally resorb without treatment. Laser surgery is a treatmentoption which uses a laser beam to seal off damaged blood vessels in theretina. This disorder can be treated with a modulator, e.g. a gapjunction and/or connexin modulator, or pannexin or pannexin channelmodulator, as described herein.

Piretinal, Intraretinal, Subretinal or Sub-Pigment Epithelial Scar/GlialTissue or Fibrin-Like Deposits

In some aspects, the connexin modulator can prevent scarring,inflammation, and the creation of fibrin formation. Thus, the connexinmodulator can prevent piretinal, intraretinal, subretinal or sub-pigmentepithelial scar/glial tissue or fibrin-like deposits. This disorder canbe treated with a modulator, e.g. a gap junction and/or connexinmodulator, or pannexin or pannexin channel modulator, as describedherein.

Retinal Angiomatous Proliferations and Retinochoroidal Anatastamosis

The retinochoroidal anastomosis represents a communication between theretinal and choroidal circulation and is described in a subset ofpatients with neovascular age-related macular degeneration (AMD).Retinal Angiomatous Proliferations are evidenced by the presence ofanastomoses between the retinal and choroidal circulations in eyes withdisciform scars. This disorder can be treated with a modulator, e.g. agap junction and/or connexin modulator, or pannexin or pannexin channelmodulator, as described herein.

Choroidal Neovascularization (CNV)

Choroidal neovascularization (CNV) is the creation of new blood vesselsin the choroid layer of the eye. This is a common sign of thedegenerative maculopathy wet AMD (age-related macular degeneration). CNVcan occur rapidly in individuals with defects in Bruch's membrane, theinnermost layer of the choroid. It is also associated with excessiveamounts of vascular endothelial growth factor (VEGF). As in wet AMD, CNVcan also occur frequently with the rare genetic disease pseudoxanthomaelasticum and rarely with the more common optic disc drusen. CNV hasalso been associated with extreme myopia or malignant myopicdegeneration, where in choroidal neovascularization occurs primarily inthe presence of cracks within the retinal (specifically) macular tissueknown as lacquer cracks. This disorder can be treated with a modulator,e.g. a gap junction and/or connexin modulator, or pannexin or pannexinchannel modulator, as described herein.

Cystic Maculopathy

Cystic maculopathy is a cyst in or around the macula, and can be treatedwith a modulator, e.g. a gap junction and/or connexin modulator, orpannexin or pannexin channel modulator, as described herein.

Retinal Thickening

Macular thickening (edema) is swelling or thickening of the part of theretina that is responsible for central vision. In some aspects, amodulator described herein can control the chorioicapillaris breakdown,thereby preventing the thickening of the retina. Thus, the modulator cantreat retinal thickening. Retinal thickening can be treated with amodulator, e.g. a gap junction and/or connexin modulator, or pannexin orpannexin channel modulator, as described herein.

Nonexudative AMD

Nonexudative AMD (“dry AMD”) results from a gradual breakdown of theretinal pigment epithelium (RPE), the accumulation of drusen deposits,and loss of function of the overlying photoreceptors. In some aspects,as described above, the modulator, e.g., a gap junction or connexinmodulator, alone or together with a pannexin or pannexin channelmodulator, as described herein. can treat nonexudative AMD.

Retinal Scarring

Retinal scarring is the development of scar tissue on, in, or under theretina, an important structure in the back of the eye. Mild scarring maynot be a serious medical issue, but large scars can cause visiondistortion and eventual vision loss. A care provider can evaluate apatient with retinal scarring to determine the extent and provide adviceon treatment options. Treatments for this condition can be invasive, anda doctor does not want to recommend a procedure that may cause more harmthan good. Patients can develop retinal scarring for a number ofreasons, including very severe myopia, ocular histoplasmosis syndrome,and wet age-related macular degeneration. It starts with irritation tothe retina that causes inflammation and leads to changes in the tissue.If this occurs repeatedly, it can start to cause significant problemsfor the patient. It may wrinkle the surface of the retina or could causethe retina to swell. Sometimes, retinal scarring causes a retinaldetachment. In some aspects, the modulator, for example, a gap junctionand/or connexin modulator and/or pannexin or pannexin channel modulatorcan control or inhibit retinal scarring and retinal inflammation, whichcontrols the tissue changes, thereby preventing or ameliorating retinalscarring.

Ocular Hypoxia

Ocular hypoxia, including retinal hypoxia, is the potentially blindingmechanism underlying a number of sight-threatening disorders includingsome types of glaucoma, central retinal artery occlusion, ischemiccentral retinal vein thrombosis, complications of diabetic eye disease(e.g., DME), AMD, and fibrosis of the eye. Hypoxia is implicated in lossof retinal ganglion cells (RGCs) occurring in such conditions. RGC deathoccurs by apoptosis or necrosis. Hypoxia-ischemia induces the expressionof hypoxia inducible factor-1α and its target genes such as vascularendothelial growth factor (VEGF) and nitric oxide synthase (NOS).Increased production of VEGF results in disruption of the blood retinalbarrier leading to retinal edema. Enhanced expression of NOS results inincreased production of nitric oxide which may be toxic to the cellsresulting in their death. Excess glutamate release in hypoxic-ischemicconditions causes excitotoxic damage to the RGCs through activation ofionotropic and metabotropic glutamate receptors. Activation of glutamatereceptors is thought to initiate damage in the retina by a cascade ofbiochemical effects such as neuronal NOS activation and increase inintracellular Ca²⁺ is a major contributing factor to RGC loss. In theposterior segment of the eye, diabetes-associated retinal hypoxia canlead to fibrosis and traction retinal detachment, a complication ofadvanced diabetic retinopathy (DR). Under the retina, similar fibrosiscan occur subsequent to subretinal hemorrhage associated withneovascular age-related macular degeneration (AMD). A therapeuticallyeffective amount of the modulator, e.g., connexin modulator, such as aconnexin 43 modulator, is any amount effective to slow, stop or reverseocular neuropathy, or to treat any of the ocular disorders describedherein.

A gap junction and/or connexin polynucleotide or oligonucleotide may beselected, for example, from modified or unmodified connexinpolynucleotides or oligonucleotides, such as modified or unmodifiedconnexin 43 antisense polynucleotides or oligonucleotides. In someembodiments, the modified connexin antisense polynucleotides, oroligonucleotides or polynucleotides comprise mixtures of modified andunmodified nucleotides. In some aspects, the connexin 43 antisensecompound used in the methods herein is an antisense oligonucleotidecomprising naturally occurring nucleobases and an unmodifiedinternucleoside linkage.

In some aspects, gap junction, connexin and/or pannexin modulators areantagonists that inhibit and/or block gap junction, connexin and/orpannexin or that inhibit and/or block upstream agonists of gap junction,connexin and/or pannexin. In some aspects the gap junction, connexinand/or pannexin antagonists include, for example, antagonists that bindto and inhibit gap junction, connexin and/or pannexin, compounds thatinhibit expression of gap junction, connexin and/or pannexin, and/orviral vectors comprising gap junction, connexin and/or pannexininhibitors or encoding proteins or antisense polynucleotides that blockor inhibit gap junctions, connexin and/or pannexin. In some aspects,species that inhibit gap junctions, connexin and/or pannexin and/orupstream agonists of gap junction, connexin and/or pannexin can beantibodies or antibody fragments, nanobodies, peptide orpeptidomimetics, receptor fragments, recombinant fusion proteins,aptamers, small molecules, or single chain variable fragments (scFv).

The methods herein provide for treatment of intraocularpressure-associated optic neuropathy such as glaucoma, in an amountsufficient to reduce intraocular pressure. In some aspects, the pannexinmodulators and connexin modulators are useful in treating traumaassociated with elevated intraocular pressure. In some aspects, thecompositions, articles of manufacture and methods of this invention areuseful in reducing the intraocular pressure to normal levels, e.g.,below 21 mm Hg, for example, below 21, 20, or 19 mm Hg. In some aspects,for example, the connexin modulators and pannexin modulators and methodsof this invention are useful in reducing the intraocular pressure tobetween, for example, about 8 to about 21 mm Hg, to between about 10 andabout 22 mm Hg, between about 10 and 21 mm Hg, or between about 12 toabout 21 mm Hg. In some aspects the compositions and methods of thisinvention are also useful in treating glaucomatous optic neuropathy evenin the absence of high intraocular pressure. In some embodiments, theconnexin modulator is, for example, a connexin 43 modulator or aconnexin 45 modulator, preferably a connexin 43 modulator. In someaspects the connexin modulator is a modulator of Cx26, Cx30, Cx31.1,Cx36, Cx37, Cx40, Cx50, Cx57 or any other connexin in the eye or bloodvessels. In some aspects, the connexin modulator can include or excludeany of the foregoing.

Featured in this invention are connexin or pannexin antisenseoligonucleotides or polynucleotides comprising at least one unmodifiednucleotide. In one aspect, the connexin antisense oligonucleotides orpolynucleotides may comprise at least one modified nucleotide, and/orhave at least one modified internucleoside linkage, and/or at least onemodified sugar moiety. The modified internucleoside linkage may be, forexample, a phosphorothioate linkage. In some aspects, for example, theconnexin 43 polynucleotide may comprise at least one nucleotidecomprising a conformationally strained nucleotide, for example, a lockednucleic acid (LNA) or a bridged nucleic acid (BNA). The lockednucleotide may be selected, from one of the following types, forexample: 2′-O—CH2-4′ (oxy-LNA), 2′-CH2-CH2-4′ (methylene-LNA),2′-NH—CH2-4′ (amino-LNA), 2′-N(CH3)-CH2-4′ (methylamino-LNA),2′-S—CH2-4′ (thio-LNA), and 2′-Se—CH2-4′ (seleno-LNA). In some aspectsthe modified nucleotide may be a locked nucleic acid or an unlockednucleic acid. In some embodiments, the connexin antisenseoligonucleotides or polynucleotides, are, for example, connexin 43antisense oligonucleotides or polynucleotides or connexin 45 antisenseoligonucleotides or polynucleotides, preferably a connexin 43 antisenseoligonucleotides or polynucleotides. In some embodiments the connexinantisense oligonucleotides or polynucleotides, are, for example,modulators of Cx26, Cx31.1, Cx36, Cx37, Cx40, Cx50, Cx57 or any otherconnexin in the eye or blood vessels.

Also featured herein are exemplary modified or unmodified connexin 43antisense compounds comprising a nucleotide sequence or modified from anucleotide sequence selected from SEQ ID NO:1-16 and or which can beincluded or excluded from the methods, compositions, kits and articlesof manufacture of this disclosure. The polynucleotides of this inventioninclude synthesized polynucleotides having a length of less than 80nucleotides, e.g., from 12-18 to about 50-80 nucleotides, preferablyabout 30 nucleotides or less, e.g., from 12 to about 30 nucleotides, andmore preferably from about 15 to about 30 nucleotides. In one example,the polynucleotide has 30 nucleotides. The methods of this inventionfeatures, in some aspects, the use of connexin 43 antisense compounds upto 40 nucleotides in length, for example, 15 to 40 nucleotides inlength, comprising a nucleotide sequence selected from SEQ ID NO:1-17,or comprising from about 8 to 40 nucleotides of SEQ ID NO:17.

TABLE 1 List of exemplary connexin 43 antisense oligonucleotides ASNSEQ ID target ASN NO. for site code ASN ASN from 5′ name sequencesequence end SEQ ID GTAATTGCGGCAAGA NO: 1 AGAATTGTTTCTGTC SEQ IDGTAATTGCGGCAGGA NO: 2 GGAATTGTTTCTGTC SEQ ID GGCAAGAGACACCAA NO: 3AGACACTACCAGCAT 24501 SEQ ID ACCCATGTTGCCT 237-256 NO: 5 GGGCACC 30004SEQ ID GTAGGCTTGAACC 281-300 NO: 6 TTGTCAA 37503 SEQ ID TCTCCCCAGGCTG372-389 NO: 7 ACTCAAC LP2/ SEQ ID CCAGGCTGACTCA 368-385 37501 NO: 4ACCGCTG 47001 SEQ ID CAGAAGCGCACAT 464-483 NO: 8 GAGAGAT 47002 SEQ IDGAAGCGCACATGA 462-481 NO: 9 GAGATTG 50501 SEQ ID AGTGTGGGTACAG 500-519NO: 10 ACACAAA LP3 SEQ ID CAGACACAAATAT 490-509 NO: 11 GATCTGC 50506SEQ ID ATATGATCTGCAG 481-500 NO: 12 GACCCAG 112304 SEQ ID GTAATTGCGGCAA1134-1153 NO: 13 GAAGAAT 1233 SEQ ID AGGCTGTGCATGG 1233-1252 NO: 14GAGTTAG 133704 SEQ ID CGCTGGTCCACAA 1316-1335 NO: 15 TGGCTAG 133705SEQ ID GCTGGCTCTGCT 1335-1354 NO: 16 TGAAGGTC

Table 1 lists the polynucleotide sequences of exemplary connexin 43polynucleotide modulators. When sequences such as SEQ ID NO:1-16 areprovided, they represent both modified and unmodified oligonucleotidesor polynucleotides. In some embodiments, the linkages between thenucleotides, and the structure of the sugar moiety of the nucleotidesmay be modified. In some embodiments, the internucleoside linkagebetween any two nucleotides can be a standard phosphodiester linkage. Insome embodiments, the internucleoside linkage between any twonucleotides can be a phosphorothioate linkage. For example, SEQ ID NO:1can be one of the following selected structures:GSTSASASTTGCGGCAAGAAGAATTGTTTCSTSGSTSC, wherein “s” denotes aphosphorothioate linkage between the two nucleotides. As anothernon-limiting example, SEQ ID NO:1 can be(G)(T)(A)(A)TTGCGGCAAGAAGAATTGTTTC(T)(G)(T)(C), wherein theparenthetical nucleotides have modified sugar moieties, as describedbelow.

Exemplary sequences are show in Table 2, which are useful in thetreatment of ocular diseases, disorders and conditions characterized byunwanted ZO-1 protein or ZO-1 protein activity or that would benefitfrom reduced ZO-1 protein or ZO-1 protein activity.

TABLE 2 List of exemplary ZO-1 AS ODNs. ASN Name Sequence SEQ ID NO:siRNA p53 5′-AAAACUCA SEQ ID NO: 228 UGUUCAAGACA GAAGGGU-3′ siRNA p533′-UUUUGAGUA SEQ ID NO: 229 revcompl CAAGUUCUGUCU UCCCA-5′ siRNA ZO-15′-CCAUCUGAU SEQ ID NO: 230 1681 GGUGUCCUACCU AAUU-3′ siRNA ZO-13′-GGUAGACUA SEQ ID NO: 231 1681 CCACAGGAUGGA revcompl UUAA-5′siRNA ZO-1 5′-GGGCUCUUG SEQ ID NO: 232 2137 GCUUGCUAUUCG AAUU-3′siRNA ZO-1 3′-CCCGAGAAC SEQ ID NO: 238 2137 CGAACGAUAAGC revcomplUUAA-5′ siRNA ZO-1 5′-CCUUCCACC SEQ ID NO: 239 5518 UUUAGAUAAAGA GAAA-3′siRNA ZO-1 3′-GGAAGGUGG SEQ ID NO: 240 5518 AAAUCUAUUUCU revcomplCUUU-5′

Table 2 lists the polynucleotide sequences of ZO-1 AS ODNs (antisenseoligodeoxtribonucleotides), shRNAs (small hairpin RNA molecules), andsiRNAs (small interfering RNA molecules).

ZO-1 was originally been identified at tight junctions, which form anetwork inside cells. This structure is only present at the intersectionbetween two cells at the cell-cell contact zone. ZO-1 is a 220-kDamembrane protein, which co-localizes with the transmembrane proteinsclaudins and occludin. Later, ZO-1 was demonstrated and identified atadherens junctions that zip cells together and thereby maintain cell andtissue polarity. These junctions also anchor the cytoskeleton, allowingthe formation of large complexes at the plasma membrane.

One sequence selected for synthesis of antisense polynucleotides thattarget ZO-1, 5′-CTGCTTTCTGTTGAGAGGCT-3′ (SEQ ID NO:236), corresponds tothe segment from base pair 3154-3169 in MUSZO1 accession number D143401.

In some aspects, the connexin 43 antisense compound is targeted to atleast about 8 nucleobases of a nucleic acid molecule encoding a connexinhaving a nucleobase sequence selected from SEQ ID NO:17. Thepolynucleotides and oligonucleotides, for example, connexin 43 antisensecompounds, may have from about 8 to about 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, or about 80 nucleotides ofSEQ ID NO:17, or a sequence complementary thereto, and/or the antisensepolynucleotide or oligonucleotide may contain any range of lengthsbetween any two of the recited lengths. The polynucleotides of thisinvention include synthesized polynucleotides having a length of lessthan 80 nucleotides, e.g., from 12-18 to about 50-80 nucleotides,preferably about 30 nucleotides or less, e.g., from 12 to about 30nucleotides, and more preferably from about 15 to about 30 nucleotides.In one example, the polynucleotide has 30 nucleotides. The methods ofthis invention features, in some aspects, the use of connexin 43antisense compounds up to 40 nucleotides in length, for example, 15 to40 nucleotides in length, comprising a nucleotide sequence selected fromSEQ ID NO:1-17. The methods of this invention features, in some aspects,the use of connexin 43 antisense compounds up to 40 nucleotides inlength, for example, 15 to 40 nucleotides in length, comprising anucleotide sequence selected from SEQ ID NO:4-17.

Human Cx 43, □ 1 LOCUS NM_000165 3088 bp mRNA linear PRI 26 OCT. 2004DEFINITION Homo sapiens gap junction protein, alpha 1,43 kDa (connexin 43) (GJA1), mRNA. (SEQ ID NO: 17)   1 acaaaaaagc ttttacgagg tatcagcact tttctttcat tagggggaag gcgtgaggaa  61 agtaccaaac agcagcggag ttttaaactt taaatagaca ggtctgagtg cctgaacttg 121 ccttttcatt ttacttcatc ctccaaggag ttcaatcact tggcgtgact tcactacttt 181 taagcaaaag agtggtgccc aggcaacatg ggtgactgga gcgccttagg caaactcctt 241 gacaaggttc aagcctactc aactgctgga gggaaggtgt ggctgtcagt acttttcatt 301 ttccgaatcc tgctgctggg gacagcggtt gagtcagcct ggggagatga gcagtctgcc 361 tttcgttgta acactcagca acctggttgt gaaaatgtct gctatgacaa gtctttccca 421 atctctcatg tgcgcttctg ggtcctgcag atcatatttg tgtctgtacc cacactcttg 481 tacctggctc atgtgttcta tgtgatgcga aaggaagaga aactgaacaa gaaagaggaa 541 gaactcaagg ttgcccaaac tgatggtgtc aatgtggaca tgcacttgaa gcagattgag 601 ataaagaagt tcaagtacgg tattgaagag catggtaagg tgaaaatgcg aggggggttg 661 ctgcgaacct acatcatcag tatcctcttc aagtctatct ttgaggtggc cttcttgctg 721 atccagtggt acatctatgg attcagcttg agtgctgttt acacttgcaa aagagatccc 781 tgcccacatc aggtggactg tttcctctct cgccccacgg agaaaaccat cttcatcatc 841 ttcatgctgg tggtgtcctt ggtgtccctg gccttgaata tcattgaact cttctatgtt 901 ttcttcaagg gcgttaagga tcgggttaag ggaaagagcg acccttacca tgcgaccagt 961 ggtgcgctga gccctgccaa agactgtggg tctcaaaaat atgcttattt caatggctgc1021 tcctcaccaa ccgctcccct ctcgcctatg tctcctcctg ggtacaagct ggttactggc1081 gacagaaaca attcttcttg ccgcaattac aacaagcaag caagtgagca aaactgggct1141 aattacagtg cagaacaaaa tcgaatgggg caggcgggaa gcaccatctc taactcccat1201 gcacagcctt ttgatttccc cgatgataac cagaattcta aaaaactagc tgctggacat1261 gaattacagc cactagccat tgtggaccag cgaccttcaa gcagagccag cagtcgtgcc1321 agcagcagac ctcggcctga tgacctggag atctagatac aggcttgaaa gcatcaagat1381 tccactcaat tgtggagaag aaaaaaggtg ctgtagaaag tgcaccaggt gttaattttg1441 atccggtgga ggtggtactc aacagcctta ttcatgaggc ttagaaaaca caaagacatt1501 agaataccta ggttcactgg gggtgtatgg ggtagatggg tggagaggga ggggataaga1561 gaggtgcatg ttggtattta aagtagtgga ttcaaagaac ttagattata aataagagtt1621 ccattaggtg atacatagat aagggctttt tctccccgca aacaccccta agaatggttc1681 tgtgtatgtg aatgagcggg tggtaattgt ggctaaatat ttttgtttta ccaagaaact1741 gaaataattc tggccaggaa taaatacttc ctgaacatct taggtctttt caacaagaaa1801 aagacagagg attgtcctta agtccctgct aaaacattcc attgttaaaa tttgcacttt1861 gaaggtaagc tttctaggcc tgaccctcca ggtgtcaatg gacttgtgct actatatttt1921 tttattcttg gtatcagttt aaaattcaga caaggcccac agaataagat tttccatgca1981 tttgcaaata cgtatattct ttttccatcc acttgcacaa tatcattacc atcacttttt2041 catcattcct cagctactac tcacattcat ttaatggttt ctgtaaacat ttttaagaca2101 gttgggatgt cacttaacat tttttttttt tgagctaaag tcagggaatc aagccatgct2161 taatatttaa caatcactta tatgtgtgtc gaagagtttg ttttgtttgt catgtattgg2221 tacaagcaga tacagtataa actcacaaac acagatttga aaataatgca catatggtgt2281 tcaaatttga acctttctca tggatttttg tggtgtgggc caatatggtg tttacattat2341 ataattcctg ctgtggcaag taaagcacac tttttttttc tcctaaaatg tttttccctg2401 tgtatcctat tatggatact ggttttgtta attatgattc tttattttct ctcctttttt2461 taggatatag cagtaatgct attactgaaa tgaatttcct ttttctgaaa tgtaatcatt2521 gatgcttgaa tgatagaatt ttagtactgt aaacaggctt tagtcattaa tgtgagagac2581 ttagaaaaaa tgcttagagt ggactattaa atgtgcctaa atgaattttg cagtaactgg2641 tattcttggg ttttcctact taatacacag taattcagaa cttgtattct attatgagtt2701 tagcagtctt ttggagtgac cagcaacttt gatgtttgca ctaagatttt atttggaatg2761 caagagaggt tgaaagagga ttcagtagta cacatacaac taatttattt gaactatatg2821 ttgaagacat ctaccagttt ctccaaatgc cttttttaaa actcatcaca gaagattggt2881 gaaaatgctg agtatgacac ttttcttctt gcatgcatgt cagctacata aacagttttg2941 tacaatgaaa attactaatt tgtttgacat tccatgttaa actacggtca tgttcagctt3001 cattgcatgt aatgtagacc tagtccatca gatcatgtgt tctggagagt gttctttatt3061 caataaagtt ttaatttagt ataaacat //

In some embodiments, the sugar moiety can be a modified sugar moiety. Insome embodiments, the modified sugar moiety can be a sugar moiety whichis a conformationally-strained sugar. In some embodiments, theconformationally-strained sugar can be a locked nucleotide (lockednucleic acid, or LNA). In some embodiments, the locked nucleotide can beselected from one of the following types: 2′-O—CH₂-4′ (oxy-LNA),2′-CH₂—CH₂-4′ (methylene-LNA), 2′-NH—CH₂-4′ (amino-LNA),2′-N(CH₃)—CH₂-4′ (methylamino-LNA), 2′-S—CH₂-4′ (thio-LNA), and2′-Se—CH₂-4′ (seleno-LNA). In some embodiments, theconformationally-strained sugar can be a bridged nucleic acid. (BNA).

As shown in Formula III, the conformationally-strained sugar can be alocked nucleic acid. In some aspects, the sugar moiety of the nucleosidecompounds can be a ribofuranose. Accordingly, a particularly suitablesubstituent X is oxygen. However, other various alternative sugarmoieties are also appropriate, and it is generally contemplated thatmodified sugars and carbocyclic moieties are also considered suitablefor use herein. Accordingly, X may also include an atom or group otherthan oxygen, and especially contemplated alternative groups X include Swhere the sugar is a sulfur sugar, CH₂, C═O, C═CH₂ or a covalent bondwhere the sugar is a carbocyclic compound, and NR (with R selected fromthe group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, andacyl) where the sugar is an amino sugar. The groups Y and Z may alsovary. In some aspects, substituents Y and Z can be selected from thegroup consisting of: O, S, CH₂, NR, C═O, C═CH₂ or a covalent bond,wherein R is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, aryl, and acyl. In some embodiments, Q may be selectedfrom none, O, S, NHR, or CH₂, wherein R is selected from the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, and acyl.

In some embodiments, B can be a base selected from one of the followingtypes of bases: substituted and unsubstituted deazapurines, azapurines,deazapyrimidines, purines, pyrimidines, and azapyrimidines. The term“substituted” as used herein refers to addition of one or morefunctional groups, wherein particularly contemplated functional groupsinclude nucleophilic (eg.: —NH₂, —OH, —SH, and —NC) and electrophilicgroups (eg.: C(O)OR, and C(X)OH), polar groups, non-polar groups (eg.:aryl, alkyl, alkenyl, or alkynyl), ionic groups (eg.: —NH₃ ⁺), andhalogens (—F, —Cl, —I, —Br), and all chemically reasonable combinationsthereof. In some aspects, B is selected from one of the following:Adenosine, Thymine, Uracil, Guanine, and Cytosine.

The groups R₁, R₂, and R₃ may be selected from the group consisting ofnone, —H, —OH, —OCH₃, alkyl, and especially methyl, —O-acyl, —N₃, —CN,and halogen. Furthermore, where contemplated nucleoside analogs includea phosphate, phosphonate group, or phosphorothioate group, it iscontemplated that especially suitable R₁ and/or R₂ groups include amonophosphate, diphosphate, triphosphate, monophosphonate,diphosphonate, triphosphonate, phosphorothioate, an amino acid esterwith a sugar OH group, or a pro-drug of the monophosphate, diphosphate,triphosphate, monophosphonate, diphosphonate, triphosphonate, orphosphorothioate.

As shown in Formula IV, the structure of the locked nucleic acid canfurther comprise Z as Oxygen (“O”), Y is CH₂, Q is none, and R₃ is none.The groups B, R₁, and R₂ can be selected from the groups as describedabove.

For any of the Markush groups set forth above, in some embodiments, eachgroup can include or exclude any of the species listed for that group.

As shown in Formula V, the structure of the conformationally-strainednucleotide can be a bridged nucleic acid (BNA). The groups X, Y, Z, R₁,R₂, R₃, and B can be selected from the groups as described above.

In some embodiments, the connexin 43 modulators can comprise peptides.The peptide sequence can comprise, for example, one or more of thefollowing sequences: SRPTEKT “Mod3”, (SEQ ID NO:173), “Peptide 1”ADCFLSRPTEKT (SEQ ID NO: 125), “Peptide 2” VACFLSRPTEKT (SEQ ID NO:126), “Peptide 11” VDCFLSRPTAKT (SEQ ID NO: 127), “Peptide 12”VDCFLSRPTEAT (SEQ ID NO: 128), “Peptide 5” VDCFLSRPTEKT (SEQ ID NO:168), “Mod1” CFLSRPTEKT (SEQ ID NO: 171), “Mod2” LSRPTEKT (SEQ ID NO:172). In some embodiments, the Carboxy-terminus can be modified. In someaspects, the carboxy-terminus modification can comprise n-alkyl chainswhich can optionally be further linked to hydrogen or other moieties. Insome embodiments, the connexin 43 peptides can include or exclude any ofthe peptides listed above or disclosed herein.

In some aspects of this invention, the connexin 43 antisenseoligonucleotide or polynucleotide has at least about 80%, 85%, 90%, 95%,97%, 98%, or 99% homology to a polynucleotide having a sequence selectedfrom SEQ ID NOs: 1 to 17 and 19.

Also featured herein are modified or unmodified connexin 45 antisensepolynucleotides comprising from 8 to about 80 nucleotides of SEQ. ID.NO:217 and modified and unmodified pannexin antisense polynucleotidescomprising from 8 to about 80 nucleotides of SEQ. ID. NO:113. Thepolynucleotides of this invention include synthesized polynucleotideshaving a length of less than 80 nucleotides, e.g., from 12-18 to about50-80 nucleotides, preferably about 30 nucleotides or less, e.g., from12 to about 30 nucleotides, and more preferably from about 15 to about30 nucleotides. In one example, the polynucleotide has 30 nucleotides.The methods of this invention features, in some aspects, the use ofconnexin 45 antisense compounds up to 40 nucleotides in length, forexample, 15 to 40 nucleotides in length, for example, comprising fromabout 8 to about 40 or from about 15 to about 40 nucleotides of SEQ IDNO:217. The methods of this invention features, in some aspects, the useof pannexin antisense compounds up to 40 nucleotides in length, forexample, 15 to 40 nucleotides in length, for example, comprising fromabout 8 to about 40 or from about 15 to about 40 nucleotides of SEQ IDNO: 117-121. In some embodiments the connexin 45 or pannexin antisensecompounds may be modified by substituting one or more uridinenucleotides residues for one or more thymine nucleotides in SEQ IDNO:217, or SEQ ID NO: 117-121.

Human Cx45, α7 LOCUS NM_005497 1191 bp mRNA linear PRI 23 DEC. 2003DEFINITION Homo sapiens gap junction protein,alpha 7, 45kDa (connexin 45) (GJA7), mRNA. (SEQ ID NO: 217)   1 atgagttgga gctttctgac tcgcctgcta     gaggagattc acaaccattc cacatttgtg  61 gggaagatct ggctcactgt tctgattgtc     ttccggatcg tccttacagc tgtaggagga 121 gaatccatct attacgatga gcaaagcaaa     tttgtgtgca acacagaaca gccgggctgt 181 gagaatgtct gttatgatgc gtttgcacct     ctctcccatg tacgcttctg ggtgttccag 241 atcatcctgg tggcaactcc ctctgtgatg     tacctgggct atgctatcca caagattgcc 301 aaaatggagc acggtgaagc agacaagaag     gcagctcgga gcaagcccta tgcaatgcgc 361 tggaaacaac accgggctct ggaagaaacg     gaggaggaca acgaagagga tcctatgatg 421 tatccagaga tggagttaga aagtgataag     gaaaataaag agcagagcca acccaaacct 481 aagcatgatg gccgacgacg gattcgggaa     gatgggctca tgaaaatcta tgtgctgcag 541 ttgctggcaa ggaccgtgtt tgaggtgggt     tttctgatag ggcagtattt tctgtatggc 601 ttccaagtcc acccgtttta tgtgtgcagc     agacttcctt gtcctcataa gatagactgc 661 tttatttcta gacccactga aaagaccatc     ttccttctga taatgtatgg tgttacaggc 721 ctttgcctct tgcttaacat ttgggagatg     cttcatttag ggtttgggac cattcgagac 781 tcactaaaca gtaaaaggag ggaacttgag     gatccgggtg cttataatta tcctttcact 841 tggaatacac catctgctcc ccctggctat     aacattgctg tcaaaccaga tcaaatccag 901 tacaccgaac tgtccaatgc taagatcgcc     tacaagcaaa acaaggccaa cacagcccag 961 gaacagcagt atggcagcca tgaggagaac     ctcccagctg acctggaggc tctgcagcgg1021 gagatcagga tggctcagga acgcttggat     ctggcagttc aggcctacag tcaccaaaac1081 aaccctcatg gtccccggga gaagaaggcc     aaagtggggt ccaaagctgg gtccaacaaa1141 agcactgcca gtagcaaatc aggggatggg      aagaactctg tctggattta a //

In some aspects of this invention, the connexin 43 antisenseoligonucleotide or polynucleotide has at least about 80%, 85%, 90%, 95%,97%, 98%, or 99% homology to a polynucleotide having a sequence selectedfrom SEQ ID NOs: 1 to 17. Connexin or pannexin modulators that areoligonucleotides or polynucleotides may have at least about 80%, 85%,90%, 95%, 97%, 98%, or 99% homology to an 8 to 80 nucleotide portion oftheir respective sequences. For example, connexin 45 modulators that areoligonucleotides or polynucleotides may have at least about 80%, 85%,90%, 95%, 97%, 98%, or 99% homology to an 8 to 80 nucleotide portion ofSEQ ID NO:217, while pannexin modulators that are oligonucleotides orpolynucleotides may have at least about 80%, 85%, 90%, 95%, 97%, 98%, or99% homology to an 8 to 80 nucleotide portion of SEQ ID NO: 117 (Panx1polynucleotide), (Panx1 polynucleotide RefSeq ID NM_015368.3), SEQ IDNO:118 (Panx2 polynucleotide), (Panx2 polynucleotide RefSeq IDNM_052839.3 for variant 1), SEQ ID NO:119 (RefSeq ID NM_001160300.1 forPanx2 polynucleotide variant 2), SEQ ID NO: 120 (RefSeq ID NR_027691.1for Panx2 polynucleotide variant 3), or SEQ ID NO: 121 (Panx3polynucleotide) (Panx3 polynucleotide RefSeq ID NM_052959.2).

In some aspects, the pannexin modulators can include or exclude pannexinpeptide sequences. The pannexin peptide sequences can comprise 8-40consecutive amino acids, an extracellular domain, an intracellulardomain, a carboxy terminus part, or an amino terminus part, of thepolypeptides SEQ ID NO: 122, Panx1 peptide), SEQ ID NO: 123, Panx2peptide), or SEQ ID NO: 124, Panx3 peptide).

In other embodiments, the connexin modulators are connexin peptides orpeptidomimetics, sometimes referred to anti-connexin peptides orpeptdomimetics, e.g., anti-connexin hemichannel blocking peptides orpeptidomimetics, for example, modified or unmodified peptides orpeptidomimentics comprising connexin extracellular domains,transmembrane regions, and connexin carboxy-terminal peptides). Theanti-connexin hemichannel blocking peptides or peptidomimetics may bemodified or unmodified. The anti-connexin hemichannel blocking peptidesor peptidomimetics are made chemically, synthetically, or otherwisemanufactured. In some embodiments, the connexin modulators are .connexin43 peptides or peptidomimetics. In some aspects, the therapeuticallyeffective modified or unmodified peptide or peptidomimetic comprises aportion of an extracellular or transmembrane domain of a connexin, suchas connexin 43 or connexin 45. In some aspects peptide or peptidomimeticcomprises a portion of an extracellular or transmembrane domain ofconnexin Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx50, Cx57 or any otherconnexin in the eye or blood vessels. In other embodiments, themodulators are pannexin peptides or peptidomimetics, sometimes referredto anti-pannexin peptides or peptdomimetics, for example, modified orunmodified peptides or peptidomimentics. In other embodiments, themodulators are pannexin peptides or peptidomimetics, sometimes referredto anti-pannexin pannexin or peptdomimetics, for example, modified orunmodified peptides or peptidomimetics. In some aspects, the connexinmodulators can include or exclude any of the foregoing.

In some embodiments the connexin modulators of this invention includeanti-connexin 43 peptides or peptidomimetics, for example, any of thepeptides described herein, including peptides comprising a portion of anextracellular domain of a connexin, and peptides comprising a portion ofa carboxy-terminal portion of a connexin useful in the methods of thisinvention, which is therapeutically effective, for example, effectivefor healing any of the neuropathic ocular disorders described herein. Insome aspects, the therapeutically effective modified or unmodifiedpeptide or peptidomimetic comprises a portion of an extracellular domainof a connexin, such as connexin 43 or connexin 45, preferably connexin43. The protein sequence of connexin 43 is shown below.

Connexin 43 (SEQ ID NO.: 19)Met Gly Asp Trp Ser Ala Leu Gly Lys Leu Leu Asp Lys Val Gln Ala1        5            10          15Tyr Ser Thr Ala Gly Gly Lys Val Trp Leu Ser Val Leu Phe Ile Phe        20          25          30Arg Ile Leu Leu Leu Gly Thr Ala Val Glu Ser Ala Trp Gly Asp Glu    35            40          45Gln Ser Ala Phe Arg Cys Asn Thr Gln Gln Pro Gly Cys Glu Asn Val  50          55           60Cys Tyr Asp Lys Ser Phe Pro Ile Ser His Val Arg Phe Trp Val Leu65          70           75           80Gln Ile Ile Phe Val Ser Val Pro Thr Leu Leu Tyr Leu Ala His Val          85           90            95Phe Tyr Val Met Arg Lys Glu Glu Lys Leu Asn Lys Lys Glu Glu Glu      100           105           110Leu Lys Val Ala Gln Thr Asp Gly Val Asn Val Asp Met His Leu Lys   115            120            125Gln Ile Glu Ile Lys Lys Phe Lys Tyr Gly Ile Glu Glu His Gly Lys 130            135           140Val Lys Met Arg Gly Gly Leu Leu Arg Thr Tyr Ile Ile Ser Ile Leu145           150         155         160Phe Lys Ser Ile Phe Glu Val Ala Phe Leu Leu Ile Gln Trp Tyr Ile          165         170          175Tyr Gly Phe Ser Leu Ser Ala Val Tyr Thr Cys Lys Arg Asp Pro Cys       180         185           190Pro His Gln Val Asp Cys Phe Leu Ser Arg Pro Thr Glu Lys Thr Ile     195          200            205Phe Ile Ile Phe Met Leu Val Val Ser Leu Val Ser Leu Ala Leu Asn   210           215         220Ile Ile Glu Leu Phe Tyr Val Phe Phe Lys Gly Val Lys Asp Arg Val225            230          235           240Lys Gly Lys Ser Asp Pro Tyr His Ala Thr Ser Gly Ala Leu Ser Pro           245         250             255Ala Lys Asp Cys Gly Ser Gln Lys Tyr Ala Tyr Phe Asn Gly Cys Ser       260           265           270Ser Pro Thr Ala Pro Leu Ser Pro Met Ser Pro Pro Gly Tyr Lys Leu     275          280            285Val Thr Gly Asp Arg Asn Asn Ser Ser Cys Arg Asn Tyr Asn Lys Gln  290          295            300Ala Ser Glu Gln Asn Trp Ala Asn Tyr Ser Ala Glu Gln Asn Arg Met305          310            315         320Gly Gln Ala Gly Ser Thr Ile Ser Asn Ser His Ala Gln Pro Phe Asp         325           330           335Phe Pro Asp Asp Asn Gln Asn Ser Lys Lys Leu Ala Ala Gly His Glu       340         345           350Leu Gln Pro Leu Ala Ile Val Asp Gln Arg Pro Ser Ser Arg Ala Ser    355          360            365Ser Arg Ala Ser Ser Arg Pro Arg Pro Asp Asp Leu Glu Ile  370           375           380

Table 3 shows extracellular loops for connexin 43 and connexin 45. Insome embodiments, the therapeutically effective modified or unmodifiedpeptide or peptidomimetic comprises a portion of the E2 extracellulardomain of a connexin, such as connexin 43 or connexin 45, preferablyconnexin 43. In some embodiments, the therapeutically effective modifiedor unmodified peptide or peptidomimetic comprises a portion of theC-terminal domain of a connexin, such as connexin 43 or connexin 45,preferably connexin 43. If a peptide or peptidomimetic modulatorcomprises a portion of an intracellular domain of a connexin, thepeptide may, in some embodiments, be conjugated to a cellinternalization transporter and may, in some instances, block zonaoccludens (ZO-1) binding to connexin 43.

TABLE 3 Extracellular loops for connexin 43 and connexin 45 E1 huCxn43ESAWGDEQSAFRCNTQQPGCENVCYDKSFPISHVR (SEQ ID NO: 129) huCx45GESIYYDEQSKFVCNTEQPGCENVCYDAFAPLSHVR (SEQ ID NO: 130) E2 huCxn43LLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKT (SEQ ID NO: 131) huCx45LIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKT (SEQ ID NO: 132)

Sequences of the E2 domain of different connexin isotypes are shown withamino acids homologous to peptide shown in bold in Table 4. Note thatlast 4 amino acids of peptide are part of the fourth membrane domain.

Table 4 provides the extracellular domain for connexin family memberswhich are used to develop peptide inhibitors described herein. Thepeptides and provided in Table 4, and fragments thereof, are used aspeptide inhibitors in certain non-limiting embodiments. In othernon-limiting embodiments, peptides comprising from about 8 to about 15,of from about 11 to about 13 amino contiguous amino of the peptides inthis Table are peptide inhibitors of the invention. In otherembodiments, conservative amino acid changes are made to the peptides orfragments thereof.

TABLE 4 Extracellular domains peptide VDCFLSRPTEKT (SEQ ID NO: 168)peptide SRPTEKTIFII (SEQ ID NO: 169) huCxn43 LLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTIFII (SEQ ID NO: 115) huCx45 LIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKTIFLL (SEQ ID NO: 116)

Other peptide sequences known to inhibit the interconnexin binding thatcan regulate connexin activity are the cytoplasmic loop of connexin 43(amino acids 119-144) L2 peptide and subparts of the L2 peptide ofconnexin 43. In some embodiments, these peptides may include or exclude,for example, the nine amino acid sequence of Gap 19, KQIEIKKFK (SEQ IDNO: 133); the native Gap19 sequence, DGVNVEMHLKQIEIKKFKYGIEEHGK (SEQ IDNO: 134); the His144→Glu L2 derivative of Gap19, as reported byShibayama (Shibayama, J. et al., Biophys. J. 91, 405404063, 2006),GVNVEMHLKQIEIKKFKYGIEEQGK (SEQ ID NO: 135); the TAT-Gap19 sequence,YGRKKRRQRRRKQIEIKKFK (SEQ ID NO: 136); the SH3 binding domain,CSSPTAPLSPMSPPGYK (SEQ ID NO: 137), or subpart thereof PTAPLSPMSPP (SEQID NO: 138); the C-terminal sequence of the CT9 or CT10 peptide, with orwithout a TAT leader sequence to increase cell penetration, RPRDDEI (SEQID NO: 139), SRPRDDLEI (SEQ ID NO: 218), YGRKKRRQRRRSRPRDDEI (SEQ ID NO:216), or YGRKKRRQRRRRPRDDEI (SEQ ID NO: 219). Other peptidomimeticsequences that can be included or excluded in the compositions, methods,kits or articles of manufacture disclosed herein are those reported byDhein (Dhein, S., Naunyn-Schmiedebergs Arch. Pharm., 350: 174-184,1994); the AAP10 peptide, H₂N-Gly-Ala-Gly-4Hyp-Pro Tyr-CONH₂ (SEQ ID NO:220), and the ZP123 peptide (rotigapeptide),Ac-D-Tyr-Pro-D-4Hyp-Gly-D-Ala-Gly-NH₂ (SEQ ID NO: 221), (Dhein, S., etal. Cell Commun. Adhes. 10, 371-378, 2013). Rotigapeptide is comprisedof the D-form of the peptides for enhanced efficacy over the nativeL-form of the peptide.

Examples of anti-pannexin agents are anti-pannexin polynucleotides,including the anti-pannexin antisense oligodeoxynucleotides (“ODN”)described below. Examples of anti-pannexin polynucleotides includeanti-pannexin oligodeoxynucleotides, including antisense (includingmodified and unmodified backbone antisense), RNAi, and miRNA and siRNA.Suitable anti-pannexin peptides include peptides that bind pannexinextracellular domains, for example, or pannexin intracellular domains.Suitable anti-pannexin agents include, for example, antisense ODNs,peptides, and peptidomimetics against Panx1. Included peptides orpeptidomimetics are anti-pannexin peptides or peptidomimetics, e.g.,pannexin complex blocking peptides (for example, anti-pannexinantibodies and antibody binding fragments) or peptidomimetics (forexample, peptidometics directed against one or more extracellular orintracellular regions of pannexin). Peptidomimetics may be complexed toone or more other agents, for example, antennapedia in order tofacilitate membrane transport for binding to intracellular pannexinregions and domains.

The terms “peptide,” “peptidomimetic” and “mimetic” include synthetic orgenetically engineered chemical compounds that may have substantiallythe same structural and functional characteristics of protein regionswhich they mimic. In the case of connexins, these may mimic, forexample, the extracellular loops of opposing connexins involved inconnexon-connexon docking and cell-cell channel formation, and/or theextracellular loops of hemichannel connexins.

As used herein, the term “peptide analogs” refer to the compounds withproperties analogous to those of the template peptide and can benon-peptide drugs. “Peptidomimetics” (also known as peptide mimetics)which include peptide and peptide-based compounds, also include suchnon-peptide based compounds such as peptide analogs. Peptidomimeticsthat are structurally similar to therapeutically useful peptides can beused to produce an equivalent or enhanced therapeutic or prophylacticeffect. Peptides and peptidomimetics may, in some aspects, be modifiedor unmodified. Generally, peptidomimetics are structural or functionalmimics (e.g., identical or similar) to a paradigm polypeptide (i.e., apolypeptide that has a biological or pharmacological function oractivity), but can also have one or more peptide linkages optionallyreplaced by a linkage selected from the group consisting of, forexample, —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis and trans), —COCH₂—,—CH(OH)CH₂—, and —CH₂SO—. The mimetic can be either entirely composed ofnatural amino acids, synthetic chemical compounds, non-natural analoguesof amino acids, or, is a chimeric molecule of partly natural peptideamino acids and partly non-natural analogs of amino acids. The mimeticcan also comprise any amount of natural amino acid conservativesubstitutions as long as such substitutions also do not substantiallyalter mimetic activity. In the case of connexins, these can mimic, forexample, the extracellular loops of opposing connexins involved inconnexon-connexon docking and cell-cell channel formation. For example,a mimetic composition can be useful as a gap junction modulating agentif it is capable of down-regulating biological actions or activities ofconnexons, such as, for example, preventing the docking of connexons toform gap-junction-mediated cell-cell communications, or preventing theopening of connexons to expose the cell cytoplasm to the extracellularmillieu. Peptidomimetics encompass those described herein, as well asthose as may be known in the art, whether now known or later developed.Peptides and peptimimetic connexin modulators may also be modified toincrease stability, improve bioavailability and/or to increase cellmembrane permeability.

In some aspects of this invention, the connexin modulator is a peptideor peptidomimetic. Exemplary connexin 43 (Cx43) or Cx26, Cx30, Cx30.3,Cx31, Cx31.1, Cx32, Cx36, Cx37, Cx40.1, Cx43, Cx46, Cx46.6, or Cx40peptide modulators that may be included or excluded in certainembodiments of this disclosure are provided in Table 64 below (E2 and T2refer to the location of a peptide in, for example, the secondextracellular domain or the second transmembrane domain).

TABLE 64 SEQ ID NO: Identifier Sequence SEQ ID CXT 2PSSRASSRASSRPRPDDLEI NO: 140 SEQ ID CXT 1 RPRPDDLEI NO: 141 SEQ ID CXT 3RPRPDDLEV NO: 142 SEQ ID CXT 4 RPRPDDVPV NO: 143 SEQ ID CXT 5 KARSDDLSVNO: 144 SEQ ID hCx40 QKPEVPNGVSPGHRLPHGYHSD NO: 145 KRRLSKASSKARSDDLSVSEQ ID Antp/CXT 2 RQPKIWFPNRRKPWKKPSSRAS NO: 146 SRASSRPRPDDLEI SEQ IDAntp/CXT 2 RQPKIWFPNRRKPWKKPSSRAS NO: 147 SRASSRPRPDDLEI SEQ IDAntp/CXT 1 RQPKIWFPNRRKPWKKRPRPDD NO: 148 LEI SEQ ID Antp/CXT 3RQPKIWFPNRRKPWKKRPRPDD NO: 149 LEV SEQ ID Antp/CXT 4RQPKIWFPNRRKPWKKRPRPDD NO: 150 VPV SEQ ID Antp/CXT 5RQPKIWFPNRRKPWKKKARSDD NO: 151 LSV SEQ ID conservative RPKPDDIDI NO: 152Cxn43 variant SEQ ID HIV-Tat/CXT 1 GRKKRRQRPPQRPRPDDLEI NO: 153 SEQ IDPenetratin/ RQIKIWFQNRRMKWKKRPRPDD NO: 154 CXT 1 LEI SEQ IDAntp-3A/CXT 1 RQIAIWFQNRRMKWAARPRPDD NO: 155 LEI SEQ ID Tat/CXT 1RKKRRQRRRRPRPDDLEI NO: 156 SEQ ID Buforin II/ TRSSRAGLQFPVGRVHRLLRKRNO: 157 Vnrs 1 PRPDDLEI SEQ ID Transportan/ GWTLNSAGYLLGKINKALAALANO: 158 CXT 1 KKILRPRPDDLEI SEQ ID MAP/CXT 1 KLALKLALKALKAALKLARPRPNO: 159 DDLEI SEQ ID K-FGF/CXT 1 AAVALLPAVLLALLAPRPRPDD NO: 160 LEISEQ ID Ku70/CXT 1 VPMLKPMLKERPRPDDLEI NO: 161 SEQ ID Prion/CXT 1MANLGYWLLALFVTMWTDVGL NO: 162 CKKRPKPRPRPDDLEI SEQ ID pVEC/CXT 1LLIILRRRIRKQAHAHSKRPRP NO: 163 DDLEI SEQ ID Pep-1/CXT 1KETWWETWWTEWSQPKKKRKVR NO: 164 PRPDDLEI SEQ ID SynB1/CXT 1RGGRLSYSRRRFSTSTGRRPRP NO: 165 DDLEI SEQ ID Pep-7/CXT 1SDLWEMMMVSLACQYRPRPDDL NO: 166 EI SEQ ID HN-1/CXT 1TSPLNIHNGQKLRPRPDDLEI NO: 167 SEQ ID SEQ-pept5, or VDCFLSRPTEKT NO: 168Peptide 5 SEQ ID SEQ-Gap27 SRPTEKTIFII NO: 169 SEQ ID SEQ-Gap26VCYDKSFPISHVR NO: 170 SEQ ID SEQ-Modl CFLSRPTEKT NO: 171 SEQ ID SEQ-Mod2LSRPTEKT NO: 172 SEQ ID SEQ-Mod3 SRPTEKT NO: 173 SEQ ID SEQ-Mod4VDCFLSRPTE NO: 174 SEQ ID SEQ-Mod5 VDCFLSRP NO: 175 SEQ ID SEQ-Mod6VDCFLS NO: 176 SEQ ID HIV-Tat/SEQ- GRKKRRQRPPQVDCFLSRPTEK NO: 177 pept5T SEQ ID Penetratin/ RQIKIWFQNRRMKWKKVDCFLS NO: 178 SEQ-pept5 RPTEKTSEQ ID Antp-3A/ RQIAIWFQNRRMKWAAVDCFLS NO: 179 SEQ-pept5 RPTEKT SEQ IDTat/SEQ- RKKRRQRRRVDCFLSRPTEKT NO: 180 pept5 SEQ ID Buforin II/TRSSRAGLQFPVGRVHRLLRKVD NO: 181 SEQ-pept5 CFLSRPTEKT SEQ ID Transportan/GWTLNSAGYLLGKINKALAALA NO: 182 SEQ-pept5 KKILVDCFLSRPTEKT SEQ IDMAP/SEQ- KLALKLALKALKAALKLAVDCF NO: 183 pept5 LSRPTEKT SEQ ID K-FGF/AAVALLPAVLLALLAPVDCFLS NO: 184 SEQpept5 RPTEKT SEQ ID Ku70/VPMLKPMLKEVDCFLSRPTEKT NO: 185 SEQpept5 SEQ ID Prion/MANLGYWLLALFVTMWTDVGLC NO: 186 SEQpept5 KKRPKPVDCFLSRPTEKT SEQ IDpVEC/SEQ- LLIILRRRIRKQAHAHSKVDCF NO: 187 pept5 LSRPTEKT SEQ ID Pep-1/KETWWETWWTEWSQPKKKRKVV NO: 188 SEQpept5 DCFLSRPTEKT SEQ ID SynB1/RGGRLSYSRRRFSTSTGRVDCF NO: 189 SEQpept5 LSRPTEKT SEQ ID Pep-7/SDLWEMMMVSLACQYVDCFLSR NO: 190 SEQpept5 PTEKT SEQ ID HN-1/TSPLNIHNGQKLVDCFLSRPTE NO: 191 SEQpept5 KT SEQ ID SEQ M3E2 FEVAFLLIQWINO: 192 SEQ ID SEQ E2a LLIQWYIGFSL NO: 193 SEQ ID SEQ E2bSLSAVYTCKRDPCPHQ NO: 194 SEQ ID SEQ E2c SRPTEKTIFII NO: 195 SEQ IDSEQ M1E1 LGTAVESAWGDEQ NO: 196 SEQ ID SEQ E1a QSAFRCNTQQPG NO: 197SEQ ID SEQ E1b QQPGCENVCYDK NO: 198 SEQ ID SEQ E1c VCYDKSFPISHVR NO: 199SEQ ID SEQ E2d KRDPCHQVDCFLSRPTEK NO: 200 SEQ ID Peptide 1 ADCFLSRPTEKTNO: 125 SEQ ID Peptide 2 VACFLSRPTEKT NO: 126 SEQ ID Peptide 11VDCFLSRPTAKT NO: 127 SEQ ID Peptide 12 VDCFLSRPTEAT NO: 128 SEQ IDGap 19subpart KQIEIKKFK NO: 133 SEQ ID Gap 19-fullDGVNVEMHLKQIEIKKFKYGIE NO: 134 EHGK SEQ ID Gap 19-denyDGVNVEMHLKQIEIKKFKYGIE NO: 135 EQGK SEQ ID TAT-Gap19YGRKKRRQRRRKQIEIKKFK NO: 136 SEQ ID 5H3-full CSSPTAPLSPMSPPGYK NO: 137SEQ ID 5H3-subpart PTAPLSPMSPP NO: 138 SEQ ID C-terminus RPRDDEI NO: 139CT9 SEQ ID C-terminus YGRKKRRQRRRSRPRDDEI NO: 216 CT9-TAT SEQ IDC-terminus SRPRDDLEI NO: 218 CT10 SEQ ID C-terminus YGRKKRRQRRRRPRDDEINO: 219 CT10-TAT SEQ ID AAP10 H₂N-Gly-Ala-Gly-4Hyp-Pro  NO: 220Tyr-CONH₂ SEQ ID ZP123 Ac-D-Tyr-Pro-D-4Hyp- NO: 221 G1y-D-A1a-G1y-NH₂SEQ ID plsl/ RVIRVWFQNKRCKDKKVDCFLS NO: 222 SEQpept5 RPTEKT SEQ IDMGB Peptide GALFLGFLGAAGSTMGAWSQPK NO: 223 P-beta/ KKRKVVDCFLSRPTEKTSEQpept5 SEQ ID MGB Peptide GALFLAFLAAALSLMGLWSQPK NO: 224 P-alpha/KKRRVVDCFLSRPTEKT SEQpept5 SEQ ID huCx26 MYVFYVMYDGFSMQRLVKCNAW NO: 244PCPNTVDCFVSRPTEKT SEQ ID huCx30 MYVFYFLYNGYHLPWVLKCGID NO: 245PCPNLVDCFISRPTEKT SEQ ID huCx30.3 LYIFHRLYKDYDMPRVVACSVE NO: 246PCPHTVDCYISRPTEKK SEQ ID huCx31 LYLLHTLWHGFNMPRLVQCANV NO: 247APCPNIVDCYIARPTEKK SEQ ID huCx31.1 LYVFHSFYPKYILPPVVKCHAD NO: 248PCPNIVDCFISKPSEKN SEQ ID huCx32 MYVFYLLYPGYAMVRLVKCDVY NO: 249PCPNTVDCFVSRPTEKT SEQ ID huCx36 LYGWTMEPVFVCQRAPCPYLVD NO: 250CFVSRPTEKT SEQ ID huCx37 LYGWTMEPVFVCQRAPCPYLVD NO: 251 CFVSRPTEKTSEQ ID huCx40.1 GALHYFLFGFLAPKKFPCTRPP NO: 252 CTGVVDCYVSRPTEKS SEQ IDhuCx43 LLIQWYIYGFSLSAVYTCKRDP NO: 253 CPHQVDCFLSRPTEKT SEQ ID huCx46IAGQYFLYGFELKPLYRCDRWP NO: 254 CPNTVDCFISRPTEKT SEQ ID huCx46.6LVGQYLLYGFEVRPFFPCSRQP NO: 255 CPHVVDCFVSRPTEKT SEQ ID huCx40IVGQYFIYGIFLTTLHVCRRSP NO: 256 CPHPVNCYVSRPTEKN

In some embodiments the connexin 43 modulator may comprise, for example,a peptide or peptidomimetic comprising, for example SEQ ID NO: 173(SRPTEKT). The peptide or peptidomimetic may also comprise, for exampleSEQ ID NO: 168 (VDCFLSRPTEKT). The peptide may contain one or moremodified amino acids, amino acid analogs, or may be otherwise modifiedto improve bioavailability or to increase penetration across the cellmembrane. For example, SEQ ID NO: 168 may be modified to obtain SEQ IDNOS:134-139 and 216. In some aspects the peptide or peptidomimeticcomprising, for example SEQ ID NO: 173 (SRPTEKT) or SEQ ID NO: 168(VDCFLSRPTEKT) comprises from 7 to 40 amino acids or amino acidanalogues and does not comprise a C-terminal peptide. In someembodiments the peptides may also be used as promoieties.

In some aspects, the Connexin 45 modulators can be peptide orpeptidomimetics comprising portions of the Connexin 45 protein thatantagonize or inhibit or block connexin-connexin interactions. Exemplarypeptide sequences for Connexin 45 peptides and peptidomimetic modulatorsare provided in Table 63.

TABLE 63 Sequences of Connexin 45 modulator peptides or peptidomimeticsSEQ ID NO. Sequence SEQ ID NO: 72 LTAVGGESIYYDEQSKFVCNTEQPGCENVCYDAFAPLSHVRFWVFQ SEQ ID NO: 73 LTAVGGESIYYDEQS SEQ ID NO: 74DEQSKFVCNTEQP SEQ ID NO: 75 TEQPGCENVCYDA SEQ ID NO: 76 VCYDAFAPLSHVRSEQ ID NO: 77 APLSHVRFWVFQ SEQ ID NO: 78 FEVGFLIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKTIFLL SEQ ID NO: 79 FEVGFLIGQYF SEQ ID NO: 80LIGQYFLYGFQV SEQ ID NO: 81 GFQVHPFYVCSRLP SEQ ID NO: 82 SRLPCHPKIDCFSEQ ID NO: 83 IDCFISRPTEKT SEQ ID NO: 84 SRPTEKTIFLL SEQ ID NO: 85SRPTEKTIFII SEQ ID NO: 86 YVCSRLPCHP SEQ ID NO: 87 QVHPFYVCSRLSEQ ID NO: 88 FEVGFLIGQYFLY SEQ ID NO: 89 GQYFLYGFQVHP SEQ ID NO: 90GFQVHPFYVCSR SEQ ID NO: 91 AVGGESIYYDEQ SEQ ID NO: 92 YDEQSKFVCNTESEQ ID NO: 93 NTEQPGCENVCY SEQ ID NO: 94 CYDAFAPLSHVR SEQ ID NO: 95FAPLSHVRFWVF SEQ ID NO: 96 LIGQY SEQ ID NO: 97 QVHPF SEQ ID NO: 98 YVCSRSEQ ID NO: 99 SRLPC SEQ ID NO: 100 LPCHP SEQ ID NO: 101 GESIYSEQ ID NO: 102 YDEQSK SEQ ID NO: 103 SKFVCN SEQ ID NO: 104 TEQPGCENSEQ ID NO: 105 VCYDAFAP SEQ ID NO: 106 LSHVRFWVFQ SEQ ID NO: 107LIQYFLYGFQVHPF SEQ ID NO: 108 VHPFYCSRLPCHP SEQ ID NO: 109VGGESIYYDEQSKFVCNTEQPG SEQ ID NO: 110 TEQPGCENVCYDAFAPLSHVRFSEQ ID NO: 111 AFAPLSHVRFWVFQ SEQ ID NO: 112 IDCFISRPTEKTIFLLSEQ ID NO: 113 DCFISRPTEKT SEQ ID NO: 114 SRPTEKT SEQ ID NO:132LIGQYFLYGFQVHPFYVCSRLP CHPKIDCFISRPTEKT

In some embodiments the connexin 45 modulator may comprise, for example,a peptide or peptidomimetic comprising, a portion of the E2 or Cterminal domain of connexin 45, for example, comprising SEQ ID NO: 114(SRPTEKT). The peptide or peptidomimetic may also comprise, for exampleSEQ ID NO: 113 (DCFISRPTEKT). In some embodiments the peptides may onlybe 3 amino acids in length, including SRL, PCH, LCP, CHP, IYY, SKF, QPC,VCY, APL, HVR, or longer.

In some aspects, the Connexin 40 modulators can be peptide orpeptidomimetics comprising portions of the Connexin 40 protein whichantagonize or inhibit connexin-connexin interactions.

Peptide Chemistry Modifications

In certain embodiments, the connexin 43 modulator peptides of thepresent invention can be linked at the amino or carboxy terminus to acellular internalization transporter. The cellular internalizationtransporter linked to the connexin 43 modulator peptides of the presentinvention may be any internalization sequence known or newly discoveredin the art, or conservative variants thereof. Non-limiting examples ofcellular internalization transporters and sequences include Antennapediasequences, TAT, HIV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II,Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC,Pep-1, SynB1, Pep-7, HN-1, BGSC (Bis-Guanidinium-Spermidine-Cholesterol,and BGTC (BisGuanidinium-Tren-Cholesterol).

The sequences of exemplary cellular internalization peptides areprovided in Table 65 below.

TABLE 65 SEQ ID NO. Identifier Sequence SEQ ID NO: 201 ANTPRQPKIWFPNRRKPWKK SEQ ID NO: 202 HIV-TAT GRKKRRQRPPQ SEQ ID NO: 203Transportan GWTLNSAGYLLGKIN KALAALAKKIL SEQ ID NO: 204 Buforin IITRSSRAGLQFPVGRVHRLLRK SEQ ID NO: 205 Tat RKKRRQRRR SEQ ID NO: 206Penetratin RQIKIWFQNRRMKWKK SEQ ID NO: 207 MAP KLALKLALKALKAALKLASEQ ID NO: 208 K-FGF AAVALLPAVLLALLAP SEQ ID NO: 209 Ku70 VPMLKPMLKESEQ ID NO: 210 Prion MANLGYWLLALFVTMW TDVGLCKKRPKP SEQ ID NO: 211 pVECLLIILRRRIRKQAHAHSK SEQ ID NO: 212 Pep-1 KETWWETWWTEWSQPKKKRRVSEQ ID NO: 213 SynB1 RGGRLSYSRRRFSTSTGR SEQ ID NO: 214 Pep-7SDLWEMMMVSLACQY SEQ ID NO: 215 HN-1 TSPLNIHNGQKL SEQ ID NO: 225 plslRVIRVWFQNKRCKDKK SEQ ID NO: 226 MGB Peptide GALFLGFLGAAGST PbetaMGAWSQPKKKRKV SEQ ID NO: 227 MGB Peptide GALFLAFLAAALSL PalphaMGLWSQPKKKRRV

Table 65 lists sequences of exemplary cellular internalizationtransporters.

In some embodiments, the connexin, pannexin and/or pannexin modulatorpeptide is fused to a transport peptide to increase the penetration intothe target cell. In some embodiments, the transport peptide can be partof a viral coating for cell penetration. In some embodiments, thetransport peptide can be fused to the connexin and/or pannexin modulatorpeptide at the carboxy or amino terminus. The transport peptide can beselected from one of the following peptides: ANTP, HIV-TAT, Transportan,Buforin II, Tat, Penetratin, MAP, K-FGF, Ku70, Prion, pVEC, Pep-1,SynB1, Pep-7, RGD, or HN-1. In some embodiments, the connexin, pannexinand/or pannexin modulator peptide can include or exclude any of theforegoing.

In one embodiment of the present invention, the amino acid sequence ofthe connexin 43 modulator peptides can be selected from the groupconsisting of any peptide SEQ ID listed herein, or a conservativevariant thereof. In a further embodiment of the present invention, theconnexin 43 modulator peptides can comprise the amino acid sequence ofSEQ ID NO: 140-200. In another embodiment of the present invention, theconnexin 43 modulator peptide further comprises a cellularinternalization transporter. In a further embodiment, the connexin 43modulator peptide can be linked at the amino terminus to the cellularinternalization transporter.

When specific proteins are referred to herein, derivatives, variants,and fragments are contemplated. Protein derivatives and variants arewell understood to those of skill in the art and can involve amino acidsequence modifications. For example, amino acid sequence modificationscan fall into one or more of three classes: insertional, substitutionalor deletional variants. Insertions include amino and/or carboxylterminal fusions as well as intrasequence insertions of single ormultiple amino acid residues. Insertions can be smaller insertions thanthose of amino or carboxyl terminal fusions, for example, on the orderof one to four residues. Deletions are characterized by the removal ofone or more amino acid residues from the protein sequence(s).Substitutions, deletions, insertions or any combination thereof may becombined to arrive at a final construct. Substitutional variants arethose in which at least one residue has been removed and a differentresidue inserted in its place. Such substitutions are referred to asconservative substitutions. The replacement of one amino acid residuewith another that is biologically and/or chemically similar is known tothose skilled in the art as a conservative substitution. A conservativesubstitution could replace one hydrophobic residue for another, or onepolar residue for another. Conservatively substituted variations of eachexplicitly disclosed sequence are included within the peptides providedherein. Conservative substitutions typically have little to no impact onthe biological activity of a resulting polypeptide. A conservativesubstitution can be an amino acid substitution in a peptide that doesnot substantially affect the biological function of the peptide. Apeptide can include one or more amino acid substitutions, from 2-10conservative substitutions, 2-5 conservative substitutions, or 4-9conservative substitutions.

Chemical Structure Modification

In certain embodiments, the chemical structure of the peptides orpeptidomimetics can be synthetically modified so as to increasetransfection uptake of the peptide. For example, the peptide orpeptidomimetic may be modified by conjugating the peptide to ahydrophobic compound, in some embodiments, through a linker moiety. Thehydrophobic compound may be, for example, one or more n-alkyl groups,which may be, for example, C6-C14 alkyl groups. In some embodiments, thepeptides may be conjugated at the N terminus to one or two dodecyl (C12)groups as described in Chen, Y S et al., J. Pharm. Sci., 102: 2322-2331(2013), herein incorporated by reference. In one embodiment, the peptidesequence CFLSRPTEKT (SEQ ID NO: 171) or VDCFLSRPTEKT (SEQ ID NO: 168)can be conjugated to two dodecyl groups to create a modified peptidewhich can modulate connexin 43, “C12-C12-Cxn43 MP.” (SEQ ID NO: 237) Theresulting structure is shown below.

The structure of C12-C12-Cxn43 MP (SEQ ID NO:237). R₁ and R₂ can behydrogen or alkyl groups. In some aspects, R₁═R₂=n-dodecyl chains.

Anti-Connexin Modulator Drugs

In some embodiments, the gap junction modulator can be a small molecule,that is, an anti-connexin modulator drug. In some aspects, theanti-connexin modulator drug can have the structure in Formula I, asdescribed herein.

Pannexin, Pannexin Channel Modulators and Other Gap Junction Modulators

Also useful in the methods of this invention are modulators includingpannexin and pannexin channel modulators and gap junction modulator orconnexin modulator drugs that may be used in any of the compositions,kits and methods described herein, in place of or in addition to theconnexin modulator or pannexin modulator.

The modulators of this invention for any of the uses featured herein mayalso comprise a gap junction modulator, which may inhibit or block Cx26,Cx31.1, Cx36, Cx37, Cx40, Cx50, Cx57 or any other connexin in the eye orblood vessels. The pharmaceutical compositions of this invention for anyof the uses featured herein may also comprise a pannexin modulator,which may inhibit or block, for example, pannexin channels.

Gap junction modulators include connexin modulators and gap junctiondrugs. Accordingly, connexin modulators are not necessarily gap junctiondrug modulators and not all gap junction drug modulators are connexinmodulators. Tonabersat, carabersat and the compounds of Formula I areconnexin modulators.

The gap junction drugs can be, for example, mycotoxins, glycyrrhetinicacid and glycyrrhetinic acid derivatives, phorbol esters, DDTs,triphenylmethanes, triphenylethanes, longchain alcohols, anesthetics,fatty acids amides, fenamates, quinine and quinine derivatives, 2-APBand 2-APB derivatives, polyamines, cyclodextrins, and peptides whichhave not been discussed above.

In some embodiments, the gap junction drugs can be selected from one ofthe following: carbamazepine, octanol, bisphenol-A, heptanol,4-(2-Butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid(DCPIB), carabersat, genistein, trans-resveratrol, carbenoxolone,HMG-CoA reductase inhibitor lovastatin, rotigaptide, metoprolol,forskolin, apigenin, tangeritin, halothane, ochratoxin A mycotoxin,patulin mycotoxin, okadaic acid, 18-alpha- and 18-beta-glycyrrhetinicacid, 17-beta-estradiol methyl ester, testosterone methyl ester,12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol esters,1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane, triphenylmethane,tris(4-chlorophenyl)methanol, (2-chlorophenyl)(diphenyl)methane,triphenylmethylchloride, sodium tetraphenylborate, tamoxifen,clomiphene, enflurane, oleamide, anandamide, arylaminobenzoates such asmeclofenamic acid and flufenamic acid, niflumic acid,5-nitro-2-(3-phenylpropylmino) benzoic acid (NPPB), quinine, quinidine,Mefloquine, PQ1 (the primaquine derivative of Mefloquine),2-aminophenoxyborate (2-APB), spermidine, spermine, cyclodextrinscontaining six, seven or eight glycopyranose units, Gap26 peptide(Val-Cys-Tyr-Asp-Lys-Ser-Phe-Pro-Ile-Ser-His-Val-Arg (SEQ ID NO: 170)),and Gap27 peptide (Ser-Arg-Pro-Thr-Glu-Lys-Thr-Ile-Phe-Ile-Ile (SEQ IDNO: 169)).

Any antisense molecule or shRNA that targets a region suitable todisrupt pannexin expression will be useful in the practicing theinvention. In some aspects this invention features RNA interference(RNAi) to pannexin. As is the case with the expression of many othergenes, pannexin expression can be knocked down in vivo or vitro throughthe use RNAi. A representative class of molecules that can be used forRNAi are short hairpin RNAs (shRNAs). One such anti-pannexin shRNAmolecule can be constructed as follows using the vector termedpSuper-Ncad.

Antisense and short hairpin RNAs targeted against human pannexin alsoinclude those that target specific sequences. Any antisense molecule orshRNA that targets a region suitable to disrupt pannexin expression, forexample, expression of Panx1 or Panx2 or Panx3, will be useful in thepracticing the invention. In some aspects this invention features RNAinterference (RNAi) to Panx1 or Panx2 or Panx3. As is the case with theexpression of many other genes, Panx1 or Panx2 or Panx3 expression canbe knocked down in vivo or vitro through the use RNAi. A representativeclass of molecules that can be used for RNAi are short hairpin RNAs(shRNAs). Inhibitors of Panx1 may include probenecid, mefloquine andcarbenoxolone, or an anti-peptide.

Chemical Delivery Modification

The modulators, including gap junction, connexin and/or pannexin andpannexin channel modulators, of the present invention can also beformulated into microparticle (microspheres, Mps) or nanoparticle(nanospheres, Nps) formulations, or both. Particulate ocular drugdelivery systems include nanoparticles (1 to 1,000 nm) andmicroparticles (1 to 1,000 μm), which are further categorized asnanospheres and microspheres and nanocapsules and microcaps. Innanocapsules and microcapsules, the drug particles or droplets areentrapped in a polymeric membrane. Particulate systems have theadvantage of intraocular delivery by injection, and their size andpolymer composition influence markedly their biological behavior invivo. Microspheres can remain in the vitreous for much longer periods oftime than nanospheres, therefore, microparticles act like a reservoirafter intravitreal injection. Nanoparticles diffuse rapidly and areinternalized in ocular tissues and cells of the anterior and posteriorsegment.

Pharmaceutical compositions are also provided for co-administration inthe form of a combined preparation, for example, as an admixture of twoor more modulators, e.g. gap junction, connexin and/or pannexin orpannexin channel modulators which may be modified or unmodified, forexample one or more gap junction, connexin and/or pannexin or pannexinchannel modulator polynucleotides and one or more gap junction, connexinand/or pannexin or pannexin channel modulator peptides orpeptidomimetics.

The term “a combined preparation” includes a “kit of parts” or “articleof manufacture” in the sense that the combination partners as definedabove can be dosed independently or by use of different fixedcombinations with distinguished amounts of the combination partners (a)and (b), i.e. simultaneously, separately or sequentially, whether inpharmaceutical form or dressing/matrix form or both. The parts of thekit can then, for example, be administered simultaneously orchronologically staggered, that is at different time points and withequal or different time intervals for any part of the kit of parts.

In one embodiment a combined preparation is administered, wherein two ormore separate modulator compositions are administered to a subject,wherein the first composition comprises a therapeutically effectiveamount of modulator, such as a gap junction, connexin and/or pannexinmodulator, e.g., an anti-connexin 43 polynucleotide, peptide, orpeptidomimetic, or a hemichannel closing compound, and the secondcomposition comprises a therapeutically effective amount of a secondmodulator, such as a gap junction, connexin and/or pannexin modulator orocular treatment agent, e.g., an anti-connexin 43 polynucleotide,peptide, or peptidomimetic, a hemichannel closing compound and/or anocular treatment agent. In another embodiment a third composition isadministered comprising one or more anti-connexin polynucleotides,peptides, or peptidomimetics, a hemichannel closing compound and/or anocular treatment agent.

Pharmaceutical compositions are provided for single, combined,simultaneous, separate, sequential, or sustained administration. In oneembodiment, a composition comprising one or more gap junction, connexinand/or pannexin modulator polynucleotides is administered at in or moredesired doses at one or more times. In another embodiment, a compositioncomprising one or more gap junction, connexin and/or pannexin modulatoris administered about the same time as one or more peptide orpeptidomimetic gap junction, connexin and/or pannexin modulators. Whenthe two compositions are administered at different times, they may beadministered within, for example, 30 minutes, 1 hour, 1 day, 1 week, or1 month part, or any time interval between any two of the recited timeperiods. In one embodiment, for example, a composition comprising one ormore anti-connexin polynucleotides is administered about the same timeas one or more anti-connexin peptides or peptidomimetics. In oneembodiment, a composition comprising one or more gap junction, connexinand/or pannexin modulator polynucleotides is administered within atleast about thirty minutes of one or more gap junction, connexin and/orpannexin modulator peptides. In one embodiment, a composition comprisingone or more gap junction, connexin and/or pannexin modulatorpolynucleotides is administered within at least about one hours of oneor more gap junction, connexin and/or pannexin modulator peptides orpeptidomimetics. In one embodiment, a composition comprising one or moregap junction, connexin and/or pannexin modulator polynucleotides isadministered within at least about twelve hours of one or more gapjunction, connexin and/or pannexin modulator peptides orpeptidomimetics. In one embodiment, a composition comprising one or moregap junction, connexin and/or pannexin modulator polynucleotides isadministered within at least about twenty-four hours of one or more gapjunction, connexin and/or pannexin modulator peptides orpeptidomimetics. In another embodiment the anti-connexin polynucleotideand gap junction, connexin and/or pannexin modulator peptide orpeptidomimetic are administered within about one hour of each other,within about one day of each other, or within about one week of eachother. Other embodiments include administration of one or more gapjunction, connexin and/or pannexin modulator polynucleotides and/or oneor more anti-connexin peptides or peptidomimetics (for example,anti-connexin peptides or peptidomimentics comprising connexinextracellular and/or connexin carboxy-terminal peptides useful for woundhealing), and one or more gap junction closing compounds useful forwound healing, one or more hemichannel closing compounds useful forwound healing, and/or one or more connexin carboxy-terminal polypeptidesuseful for wound healing. Modulator doses may be administered QD, BID,TID, QID, or in weekly doses, e.g., QIW, BIW QW. They may also beadministered PRN, and hora somni.

Dosage Forms and Formulations and Administration

All descriptions with respect to dosing, unless otherwise expresslystated, apply to the modulators of the invention, including gap junctionmodulators, pannexin channel modulators, connexin modulators andpannexin modulators.

The modulators, including hemichannel, gap junction, and/or pannexinmodulators, of the invention can be dosed, administered or formulated asdescribed herein.

The modulators, including hemichannel, gap junction, and/or pannexinmodulators, of the invention can be administered to a subject in need oftreatment, having an ocular neuropathy. Thus, in accordance with theinvention, there are provided formulations by which pannexin and/or aconnexin, for example, connexin 43 or connexin 45 can be modulatedand/or cell-cell communication can be downregulated in a transient andsite-specific manner.

The modulators, including gap junction, connexin and/or pannexin andpannexin channel modulators may be present in the formulation in asubstantially isolated form. It will be understood that the product maybe mixed with carriers or diluents that will not interfere with theintended purpose of the product and still be regarded as substantiallyisolated. A product of the invention may also be in a substantiallypurified form, in which case it will generally comprise about 80%, 85%,or 90%, e.g. at least about 88%, at least about 90, 95 or 98%, or atleast about 99% of a polynucleotide, for example (or other connexinmodulator such as a connexin 43 modulator) or dry mass of thepreparation.

In one embodiment, the gap junction, connexin and/or modulator is amodified or unmodified connexin 43 or 45 antisense polynucleotide oroligonucleotide, a modified or unmodified connexin peptide oranti-connexin 43 or 45 peptidomimetic. In some embodiments, the connexinmodulator can block or reduce hemichannel opening. In some embodiments,a modified or unmodified connexin peptide or peptidomimetic, isadministered to block hemichannel or gap junction function and/or toexert regulatory effects that reduce connexin expression or theformation of hemichannels or gap junctions, e.g., by downregulation ofconnexin protein expression. In some embodiments, the modified orunmodified connexin peptide or peptidomimetic, is administered prior tothe administration of a modified or unmodified anti-connexinpolynucleotide or oligonucleotide, to block hemichannel or gap junctionfunction prior to downregulation of connexin protein expression by thepolynucleotide or oligonucleotide. In some embodiments the connexinmodulator is a connexin 43 modulator.

The pharmaceutical formulations of this invention may further compriseone or more pharmaceutically acceptable excipients suitable fordelivering the modulators, including gap junction, connexin and/orpannexin and pannexin channel modulators to the eye. In some aspects,administering a pannexin modulator, or a connexin modulator (forexample, a connexin 43 modulator or connexin 45 modulator, preferably aconnexin 43 modulator), to the eye of a subject provides therapeuticallyeffective amounts of the connexin modulator or pannexin modulator to theeye or specific compartment of the eye. In some instances the gapjunction, connexin and/or, pannexin modulator may be administered bytopical, corneal, intravitreal, subconjunctival, or periocularadministration. In some aspects, administration may also beintraperitoneal administration or parenteral administration, providedthat a therapeutically effective dose contacts the eye. In some aspectsof the methods of this invention, the gap junction, connexin and/orpannexin modulator may be administered to the eye by injection, forexample, by intraocular injection, intravitreal injection or byperiocular routes including subconjunctival, retrobulbar, peribulbar,and posterior sub-Tenon injections. In some aspects, the gap junction,connexin and/or pannexin modulator may be provided to or injecteddirectly into or near the trabecular meshwork. In some aspects,sub-conjunctival administration may provide for sustained delivery,while minimizing the dose frequency. In some embodiments,subconjunctival administration may increase the bioavailabiltiy ofhydrophilic drugs because they do not have to penetrate the conjunctivalepithelium. In some embodiments, a microneedle, needle, iontophoresisdevice or implant may be used for administration of the connexinmodulator or pannexin modulator. The implant can be, for example, adissolvable disk material such as that described in S. Pflugfelder etal., ACS Nano, 9 (2), pp 1749-1758 (2015). The modulator, e.g. gapjunction, connexin and/or pannexin or pannexin channel modulator of thisinvention may also be administered at or near the trabecular meshwork orciliary body, so that the gap junction, connexin and/or pannexin orpannexin channel modulator contacts the trabeular meshwork and/or theciliary body, respectively. The modulator, e.g. gap junction, connexinand/or pannexin and/or pannexin channelmodulator may be administeredonce, or more than once. The connexin modulator may be, for example, aconnexin 43 modulator or a connexin 45 modulator, preferably be aconnexin 43 modulator. In some embodiments a microneedle may be used todeliver any of the compositions of this invention to the choroid.

For example, in some embodiments, a modulator, such as a connexin 43modulator, is administered to the subject, e.g. the eye of the subject,providing therapeutically effective amounts of the modulator to the eyeor specific compartment of the eye. In some instances the modulator,e.g. connexin 43 or pannexin 1 or pannexin 1 channel modulator, may beadministered by topical, corneal, intravitreal, subconjunctival, orperiocular administration. In some aspects, administration may also beintraperitoneal administration. In some embodiments, a microneedle,needle, iontophoresis device, or implant may be used for administrationof the modulator, e.g. connexin 43 modulator. The modulators, e.g.connexin 43 modulators, of this invention may also be administered tothe trabecular meshwork or ciliary body. In some aspects, themodulators, e.g. connexin 43 modulators of this invention may beadministered via intraventricular, and/or intrathecal, and/orextradural, and/or subdural, and/or epidural routes.

A modulator, e.g. a gap junction channel modulator, such as peptide 5,and/or an analogue or prodrug thereof, compounds of formula I, forexample tonabersat, and analogs or prodrugs of any of the foregoingcompounds, and/or a pannexin or pannexin channel modulator, e.g.,compounds of formula VI, for example probenecid and an analogues orprodrugs thereof, and/or a synthetic mimetic peptide blocker of pannexin1, e.g., ¹⁰Panx1 or an analogue or prodrug thereof may be administeredalone or in combination with one or more additional ingredients and maybe formulated into pharmaceutical compositions including one or morepharmaceutically acceptable excipients, diluents and/or carriers.

“Pharmaceutically acceptable diluents, carriers and/or excipients” isintended to include substances that are useful in preparing apharmaceutical composition, may be co-administered with compounds offormula I, for example tonabersat, and analogs of any of the foregoingcompounds, while allowing it to perform its intended function, and aregenerally safe, non-toxic and neither biologically nor otherwiseundesirable. Pharmaceutically acceptable diluents, carriers and/orexcipients include those suitable for veterinary use as well as humanpharmaceutical use. Suitable carriers and/or excipients will be readilyappreciated by persons of ordinary skill in the art, having regard tothe nature of compounds of formula I, for example tonabersat, andanalogs of any of the foregoing compounds. However, by way of example,diluents, carriers and/or excipients include solutions, solvents,dispersion media, delay agents, polymeric and lipidic agents, emulsionsand the like. By way of further example, suitable liquid carriers,especially for injectable solutions, include water, aqueous salinesolution, aqueous dextrose solution, and the like, with isotonicsolutions being preferred for intravenous, intraspinal, andintracisternal administration and vehicles such as liposomes being alsoespecially suitable for administration of agents.

Compositions may take the form of any standard known dosage formincluding tablets, pills, capsules, semisolids, powders, sustainedrelease formulation, solutions, suspensions, elixirs, aerosols, liquidsfor injection, gels, creams, transdermal delivery devices (for example,a transdermal patch), inserts such as ocular inserts, or any otherappropriate compositions. Persons of ordinary skill in the art to whichthe invention relates will readily appreciate the most appropriatedosage form having regard to the nature of the condition to be treatedand the active agent to be used without any undue experimentation. Itshould be appreciated that one or more of gap junction channelmodulator, such as peptide 5, and/or an analogue thereof, compounds offormula I, for example tonabersat, and analogs of any of the foregoingcompounds, and/or a pannexin modulator, e.g., probenecid and an analoguethereof, and/or a synthetic mimetic peptide blocker of pannexin 1, e.g.,¹⁰Panx1, or an analogue thereof may be formulated into a singlecomposition. In certain embodiments, preferred dosage forms include aninjectable solution and an oral formulation.

Compositions of the invention may contain any appropriate level ofmodulator, e.g. gap junction channel modulator, such as peptide 5,and/or an analogue thereof, compounds of formula I, for exampletonabersat, and analogs of any of the foregoing compounds, and/or apannexin modulator, e.g., probenecid and an analogue thereof, and/or asynthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1, or ananalogue thereof, having regard to the dosage form and mode ofadministration. However, by way of example, compositions of use in theinvention may contain from approximately 0.1% to approximately 99% byweight, preferably from approximately 1% to approximately 60% by weight,of compounds of formula I, for example tonabersat, and analogs of any ofthe foregoing compounds, depending on the method of administration.

In addition to standard diluents, carriers and/or excipients, acomposition in accordance with the invention may be formulated with oneor more additional constituents, or in such a manner, so as to enhancethe activity or bioavailability of gap junction channel modulator, suchas peptide 5, and/or an analogue thereof, compounds of formula I, forexample tonabersat, and analogs of any of the foregoing compounds,and/or a pannexin modulator, e.g., probenecid and an analogue thereof,and/or a synthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1,or an analogue thereof, help protect the integrity or increase thehalf-life or shelf life thereof, enable slow release upon administrationto a subject, or provide other desirable benefits, for example. Forexample, slow release vehicles include macromers, poly(ethylene glycol),hyaluronic acid, poly(vinylpyrrolidone), or a hydrogel. By way offurther example, the compositions may also include preserving agents,solubilising agents, stabilising agents, wetting agents, emulsifyingagents, sweetening agents, colouring agents, flavouring agents, coatingagents, buffers and the like. Those of skill in the art to which theinvention relates will readily identify further additives that may bedesirable for a particular purpose.

Compounds of Formula I, for example tonabersat, and analogs of any ofthe foregoing compounds, may be administered by a sustained-releasesystem. Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, poly(2-hydroxyethyl methacrylate), ethylenevinyl acetate, or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release compositions also include a liposomally entrappedcompound. Liposomes containing compounds of formula I, for exampletonabersat, and analogs of any of the foregoing compounds, may beprepared by known methods, including, for example, those described in:DE 3,218,121; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641;Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545;and EP 102,324. Ordinarily, the liposomes are of the small (from orabout 200 to 800 Angstroms) unilamellar type in which the lipid contentis greater than about 30 mole percent cholesterol, the selectedproportion being adjusted for the most efficacious therapy. Slow releasedelivery using PGLA nano- or microparticles, or in situ ion activatedgelling systems may also be used, for example.

Additionally, it is contemplated that a pharmaceutical composition inaccordance with the invention may be formulated with additional activeingredients or agents which may be of therapeutic or other benefit to asubject in particular instances. Persons of ordinary skill in the art towhich the invention relates will readily appreciate suitable additionalactive ingredients having regard to the description of the inventionherein and nature of the disorder to be treated.

The compositions may be formulated in accordance with standardtechniques as may be found in such standard references as Gennaro AR:Remington: The Science and Practice of Pharmacy, 20^(th) ed.,Lippincott, Williams & Wilkins, 2000, for example. However, by way offurther example, the information provided in US2013/0281524 or U.S. Pat.No. 5,948,811 may be used.

In certain embodiments, the invention provides a combination productcomprising (a) compounds of formula I, for example tonabersat, andanalogs of any of the foregoing compounds and (b) one or more additionalactive agent wherein the components (a) and (b) are adapted foradministration simultaneously or sequentially.

In a particular embodiment of the invention, a combination product inaccordance with the invention is used in a manner such that at least oneof the components is administered while the other component is stillhaving an effect on the subject being treated.

The gap junction channel modulator, such as peptide 5, and/or ananalogue thereof, compounds of formula I, for example tonabersat, andanalogs of any of the foregoing compounds, and/or a pannexin modulator,e.g., probenecid and an analogue thereof, and/or a synthetic mimeticpeptide blocker of pannexin 1, e.g., ¹⁰Panx1, or an analogue thereof andone or more additional active agent may be formulated in suitable formfor direct administration to a subject (for example, as an agent orpharmaceutical composition). Alternatively, the combination product maycomprise one or more pharmaceutical carrier compositions in one or moreseparate containers; the agent(s) being mixed with a one or morepharmaceutical carrier composition prior to administration.

The compounds of formula I, for example tonabersat, and analogs of anyof the foregoing compounds and one or more additional active agent maybe contained in the same or one or more different containers andadministered separately, or mixed together, in any combination, andadministered concurrently.

The combination product may also comprise additional agents andcompositions in further separate containers as may be necessary for aparticular application.

Any container suitable for storing and/or administering a pharmaceuticalcomposition may be used in a combination product of the invention.Suitable containers will be appreciated by persons skilled in the art.By way of example, such containers include vials and syringes. Thecontainers may be suitably sterilised and hermetically sealed.

Such combination products may be manufactured in accordance with themethods and principles provided herein and those known in the art.

Also provided is combination product used in a method as hereindescribed.

The pharmaceutical compositions of this invention include, for example,ocular delivery forms and formulations. Such delivery forms andformulations include those for the treatment of a subject as disclosedherein. The pharmaceutical formulations of this invention may furthercomprise one or more pharmaceutically acceptable excipients. Thepharmaceutically acceptable excipients for ocular administration may beophthalmologically acceptable excipients. In some aspects, theformulations may provide for sustained delivery of the connexinmodulator and/or ocular treatment agent to a selected segment orcompartment of the eye. The formulations may, in some aspects, providehigh ocular drug bioavailability, be safe and non-toxic, and/or havelittle systemic side effects or complications at the site ofadministration. Exemplary polynucleotide formulations for use in themethods of this invention have the ease of localized delivery, ease ofadministration and a “no side effect” profile.

In some embodiments the pharmaceutical formulations of this inventionmay comprise any of the modulators, e.g. gap junction and/or connexinmodulators or pannexin modulators described herein, for example, amodified or unmodified connexin 43 antisense oligonucleotide orpolynucleotide or a modified or unmodified connexin 43 peptide orpeptidomimetic. Connexin 43 antisense oligonucleotide that are includedin the formulation may be, in some embodiments, an unmodified connexin43 antisense oligodeoxynucleotide or a modified connexin 43 antisenseoligodeoxynucleotide. In some aspects, the pharmaceutical compositionscan include or exclude any of the foregoing.

In some aspects, administering a modulator, for example, a connexinmodulator, pannexin modulator, and/or a gap junction modulator (forexample, a connexin 43 modulator or a Cx45, Cx26, Cx30, Cx31.1, Cx36,Cx37, Cx40, Cx45, Cx50, Cx57, or any other connexin in the eye), to theeye of a subject provides therapeutically effective amounts of theconnexin modulator or pannexin modulator to the eye or specificcompartment of the eye. The connexin modulator may preferably be aconnexin 43 modulator. In some instances the pannexin modulator orconnexin modulator may be administered by topical, intravitreal,subconjunctival, or periocular administration. In some aspects,administration may also be intraperitoneal administration or parenteraladministration, provided that a therapeutically effective dose contactsthe eye. In some aspects of the methods of this invention, the connexinmodulator or pannexin modulator may be administered to the eye byinjection, for example, by intraocular injection, intravitreal injectionor by periocular routes including subconjunctival, retrobulbar,peribulbar, and posterior sub-Tenon injections. In some aspects, theconnexin modulator may be injected directly into or near the trabecularmeshwork. In some aspects, sub-conjunctival administration may providefor sustained delivery, while minimizing the dose frequency. In someembodiments, subconjunctival administration may increase thebioavailabiltiy of hydrophilic drugs because they do not have topenetrate the conjunctival epithelium. In some embodiments, amicroneedle, needle, or implant may be used for administration of theconnexin modulator. The connexin modulators of this invention may alsobe administered to the ciliary body. The connexin modulator may beadministered once, or more than once. The connexin modulator may be, forexample, any of the connexin modulators described herein. In someaspects, the connexin or pannexin modulator can include or exclude anyof the foregoing.

The connexin and pannexin channel modulators of this invention may alsobe administered to the trabecular meshwork or ciliary body.

The connexin and pannexin channel modulators of this invention may alsobe administered to the trabecular meshwork or ciliary body.

The gap junction, connexin, hemichannel, and/or pannexin modulators, forexample, connexin 43 modulators and pannexin 1 modulators may, in someaspects, be formulated to provide controlled and/or compartmentalizedrelease to the site of administration in the eye. In some aspects ofthis invention, the formulations may be immediate, or extended orsustained release dosage forms. In some aspects, the dosage forms maycomprise both an immediate release dosage form, in combination with anextended and/or sustained release dosage form. In some aspects bothimmediate and sustained and/or extended release of connexin or pannexinmodulators can be obtained by combining a modified or unmodifiedconnexin or pannexin antisense oligonucleotide or polynucleotide,together with a modified or unmodified peptide or peptidomimetic in animmediate release form. In some aspects of this invention the connexinmodulators are, for example, connexin 43 modulators or other connexinmodulators of this disclosure. In some aspects of this invention, thedosage forms may be ocular implants, for example, biodegradable ornonbiodegradable implants.

In some aspects of this invention, the caderin modulator or gap junctionand/or connexin modulator, e.g., a connexin 43 modulator, may beformulated for compartmentalized release of the modulator, for example,by adjusting the size or coating of the particles. For example, in someaspects, particle formulations of the caderin modulator or connexinmodulator, e.g., a connexin 43 modulator, can be administered for use inthe methods of this invention. Ocular drug delivery systems comprisingparticles may comprise, in some aspects, nanoparticles having a meandiameter of less than 1,000 nm, for example, 1-1000 nm, and/ormicroparticles having a mean diameter between 1 to 1,000 μm. Thenanoparticles or microparticles may be, for example, nanospheres ormicrospheres, or encapsulated nanocapsules and microcapsules, in whichthe connexin modulator is encapsulated in a polymeric coating. Theparticle formulations may also comprise liposomes. In some aspects theconnexin modulator is can include or exclude a modulator of connexin 45,Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx50, or Cx57 or any otherconnexin in the eye or blood vessels.

In some aspects, the gap junction and/or connexin modulator or pannexinmodulator can be formulated for targeted delivery to the choroid and/orretina following peripheral administration, for example, intravenous orintraperitoneal administration. In some aspects, the connexin modulatormay be, for example, a connexin 43 modulator or other connexinmodulators of this disclosure.

In some aspects, the formulation for targeted delivery to the choroidand/or retina can comprise the administering of gap junction and/orconnexin modulator or pannexin modulator infused particles that presentan ocular targeting moiety on the surface of the particles.

The invention comprises methods for modulating the function of a gapjunction channel and/or a hemichannel and for the treatment of variousdisorders. It should be appreciated that such methods may be performedin vivo, ex vivo and/or in vitro, as is appropriate. In certainembodiments, the methods may be performed for experimental and/ornon-therapeutic purposes. In certain embodiments, such methods maycomprise the step of administering gap junction channel modulator, suchas peptide 5, and/or an analogue thereof, compounds of formula I, forexample tonabersat, and analogs of any of the foregoing compounds,and/or a pannexin modulator, e.g., probenecid and an analogue thereof,and/or a synthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1,or an analogue thereof to one or more cell or a sample comprising one ormore cell in vitro or ex vivo. In other embodiments, such methods maycomprise the step of administering a gap junction channel modulator,such as peptide 5, and/or an analogue thereof, compounds of formula I,for example tonabersat, and analogs of any of the foregoing compounds,and/or a pannexin modulator, e.g., probenecid and an analogue thereof,and/or a synthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1,or an analogue thereof to a subject.

Methods of the invention comprise administering modulator, e.g. a gapjunction channel modulator, such as peptide 5, and/or an analoguethereof, compounds of formula I, for example tonabersat, and analogs ofany of the foregoing compounds, and/or a pannexin modulator, e.g.,probenecid and an analogue thereof, and/or a synthetic mimetic peptideblocker of pannexin 1, e.g., ¹⁰Panx1, or an analogue thereof alone or ina combination with one or more other agents (including for exampleactive agents) or therapies as may be desired.

Administration of a modulator, e.g. a gap junction channel modulator,such as peptide 5, and/or an analogue thereof, compounds of formula I,for example tonabersat, and analogs of any of the foregoing compounds,and/or a pannexin modulator, e.g., probenecid and an analogue thereof,and/or a synthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1,or an analogue thereof to a subject may occur by any means capable ofdelivering the agents to a target site within the body of a subject. Byway of example, gap junction channel modulator, such as peptide 5,and/or an analogue thereof, compounds of formula I, for exampletonabersat, and analogs of any of the foregoing compounds, and/or apannexin modulator, e.g., probenecid and an analogue thereof, and/or asynthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1, or ananalogue thereof may be administered by one of the following routes:oral, topical, systemic (eg. Intravenous, intra-arterial,intra-peritoneal, transdermal, intranasal, or by suppository),parenteral (eg. intramuscular, subcutaneous, or intravenous orintra-arterial injection), by implantation, and by infusion through suchdevices as osmotic pumps, transdermal patches, and the like. Skilledpersons may identify other appropriate administration routes. Exemplaryadministration routes are also outlined in: Binghe, W. and B. Wang(2005). Drug delivery: principles and applications, Binghe Wang, TerunaSiahaan, Richard Soltero, Hoboken, N.J. Wiley-Interscience, c2005. Inone embodiment, gap junction channel modulator, such as peptide 5,and/or an analogue thereof, compounds of formula I, for exampletonabersat, and analogs of any of the foregoing compounds, and/or apannexin modulator, e.g., probenecid and an analogue thereof, and/or asynthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1, or ananalogue thereof is administered systemically. In another embodiment,gap junction channel modulator, such as peptide 5, and/or an analoguethereof, compounds of formula I, for example tonabersat, and analogs ofany of the foregoing compounds, and/or a pannexin modulator, e.g.,probenecid and an analogue thereof, and/or a synthetic mimetic peptideblocker of pannexin 1, e.g., ¹⁰Panx1, or an analogue thereof isadministered orally. In another embodiment, gap junction channelmodulator, such as peptide 5, and/or an analogue thereof, compounds offormula I, for example tonabersat, and analogs of any of the foregoingcompounds, and/or a pannexin modulator, e.g., probenecid and an analoguethereof, and/or a synthetic mimetic peptide blocker of pannexin 1, e.g.,¹⁰Panx1, or an analogue thereof is administered topically.

In another embodiment, compounds of formula I, for example tonabersat,and analogs of any of the foregoing compounds is administeredsystemically, such as by intravenous, intra-arterial or intraperitonealadministration, such that the final circulating concentration is fromapproximately 0.001 to approximately 150 micromolar, or higher up to200, 300, 400, 500, 600, 700, 800, 900 or 1000 micromolar. The finalcirculating concentration can be 0.001, 0.002, 0.003, 0.004, 0.005,0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 100, 110, 120, 130, 140, or 150 micromolar, or anyconcentration between any of the two recited numbers, or higher asdescribed above and any concentration within the ranges noted. Asmentioned herein, the invention also comprises combination therapies inwhich one or more additional active agent is also administered to asubject. Skilled persons will appreciate desirable dosages for the oneor more active agent having regard to the nature of that agent and theprinciples discussed herein before.

A modulator, e.g. a gap junction channel modulator, such as peptide 5,and/or an analogue thereof, compounds of formula I, for exampletonabersat, and analogs of any of the foregoing compounds, and/or apannexin modulator, e.g., probenecid and an analogue thereof, and/or asynthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1, or ananalogue thereof may be used in the invention alone or in combinationwith one or more additional agent or composition of use in the treatmentof a particular disorder. Co-administration may allow for improvedalleviation or amelioration of one or more symptoms, reduction of thelength or extent of a disease, delay or slowing of the progression ofdisease, amelioration, palliation or stabilization of the disease state,partial or complete remission, prolonged survival and/or otherbeneficial therapeutic results. Such treatments may be administeredsimultaneously or sequentially in any order with a period of timebetween administrations. One of skill in the art will readily appreciatemethods of administering agents or therapies simultaneously orsequentially and possible time periods between administrations. Thetherapies may be administered by the same or different routes.

In certain embodiments, treatment according to the invention may involvethe administration of one or more other agents to a subject. Forexample, one or more agents of use in promoting the general health of asubject, or reducing one or more side-effects of therapy could beadministered. Skilled persons will readily appreciate various agentswhich may be beneficial to administer having regard to the disease to betreated, for example. In some embodiments where a pannexin modulator isinitially co-administered with a gap junction channel modulator, theadministration of the pannexin modulator may be stopped or tapered,while administration of the gap junction channel modulator continues. Insome embodiments a pannexin modulator may be administered immediatelyafter or shortly after an ischemic injury, while a gap junction channelmodulator may be administered post-ischemia.

Administration of a modulator, e.g. a gap junction channel modulator,such as peptide 5, and/or an analogue thereof, compounds of formula I,for example tonabersat, and analogs of any of the foregoing compounds,and/or a pannexin modulator, e.g., probenecid and an analogue thereof,and/or a synthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1,or an analogue thereof, and optionally one or more other active agent,may occur at any time during the progression of a disorder, or prior toor after the development of a disorder or one or more symptom of adisorder. In one embodiment, gap junction channel modulator, such aspeptide 5, and/or an analogue thereof, compounds of formula I, forexample tonabersat, and analogs of any of the foregoing compounds,and/or a pannexin modulator, e.g., probenecid and an analogue thereof,and/or a synthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1,or an analogue thereof is administered on a daily basis for an extendedperiod to assist with ongoing management of symptoms. In anotherembodiment, gap junction channel modulator, such as peptide 5, and/or ananalogue thereof, compounds of formula I, for example tonabersat, andanalogs of any of the foregoing compounds, and/or a pannexin modulator,e.g., probenecid and an analogue thereof, and/or a synthetic mimeticpeptide blocker of pannexin 1, e.g., ¹⁰Panx1, or an analogue thereof isadministered on a daily basis for an extended period or life-long toprevent or delay the development of a disorder.

Preferably the modulator, e.g. gap junction, connexin, and/or pannexinor pannexin channel modulators of the invention are combined with apharmaceutically acceptable carrier or diluent to produce apharmaceutical composition. The term “pharmaceutically acceptablecarrier” refers to any pharmaceutical carrier that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition, and which can be administered without undue toxicity.

“Pharmaceutically acceptable carrier” for ocular administration will beopthalmologically acceptable carriers.

Pharmaceutically acceptable salts can also be present, e.g., mineralacid salts such as hydrochlorides, hydrobromides, phosphates, sulfates,and the like; and the salts of organic acids such as citrates, acetates,propionates, malonates, benzoates, and the like.

Suitable carriers and diluents include buffered, aqueous solutions,saline, dextrose, glycerol, isotonic saline solutions, for examplephosphate-buffered saline, isotonic water, and the like and combinationsthereof. In some embodiments, carriers may include propylene glycol,dimethyl isosorbide, and water, and even more particularly, phosphatebuffered saline, isotonic water, deionized water, monofunctionalalcohols and symmetrical alcohols. In some embodiments pharmaceuticallyacceptable carrier or diluent may be or contain a thermosettingpoloxamer (which may be a liquid or gel, depending on the temperature),a carboxycellulose (e.g. carboxymethylcellulose), a collagen (e.g., aType I collagen), a collagenous material comprising tropocollagen, ahyaluronan or derived-hyaluronic acid, and/or an oil (e.g., Emu oil).Suitable carriers can be large, slowly metabolized macromolecules suchas proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, and amino acid copolymers. The pharmaceuticalcompositions of this invention do not comprise a connexin modulator insterile water as the only vehicle. In some embodiments, the formulationswill comprise pannexin or connexin modulators, for example, connexin 43modulators, for example, 43 antisense oligonucleotide that are includedin the formulation may be, in some embodiments, an unmodified ormodified connexin 43 antisense oligodeoxynucleotide.

In one aspect the reverse-thermosetting gel may be a liquid at lowtemperatures, for example at 2-8° C., and which undergoes a reversibleliquid to gel transition at temperatures greater than approximately 15°C. Thus, in some embodiments the carrier may be a liquid at temperaturesbelow approximately 15° C., but may form a gel at temperatures aboveapproximately 15° C., such as room temperature or at body temperature.In some instances, the gel is a nonionicpolyoxyethylene-polyoxypropylene copolymer gel. In some embodiments thegel is a pluronic gel. The pluronic gel may be, for example, poloxamer407, also sometimes referred to as Pluronic F-127 (BASF). In someembodiments, the formulations of this invention may comprise from about15 to about 30% (w/v) gel. In some embodiments, the formulations of thisinvention may comprise from about 20 to about 25% (w/v) gel. In someembodiments, the formulations of this invention may comprise about 22.6%(w/v) poloxamer 407 gel. Other suitable formulations include pluronicgel-based formulations, hydroxymethylcellulose formulations,hydroxyethylcellulose formulations, carboxymethylcellulose (CMC)-basedformulations, and hydroxypropylmethylcellulose (HPMC)-basedformulations. The composition may be formulated for any desired form ofdelivery, including topical, instillation, parenteral, intramuscular,subcutaneous, or transdermal administration. Other useful formulationsinclude slow or delayed release preparations.

In addition, if desired substances such as wetting or emulsifyingagents, stabilizing or pH buffering agents, or preservatives may also bepresent. In some embodiments, the pharmaceutical compositions of thisinvention will comprise suitable ophthalmically acceptable buffers, suchas acetate buffers, citrate buffers, phosphate buffers, borate buffersand mixtures thereof. In some embodiments, the buffers useful in thepresent invention include boric acid, sodium borate, sodium phosphates,including mono, di- and tri-basic phosphates, such as sodium phosphatemonobasic monohydrate and sodium phosphate dibasic heptahydrate, andmixtures thereof. In some embodiments, the preservative may bestabilized chlorine dioxide, cationic polymers or quaternary ammoniumcompounds. In some embodiments the pharmaceutical compositions may alsocomprise wetting agents, nutrients, viscosity builders, antioxidants,and the like, for example, disodium ethylene diamine tetraacetate,alkali metal hexametaphosphate, citric acid, sodium citrate, sodiummetabisulfite, sodium thiosulfate, N-acetylcysteine, butylatedhydroxyanisole, butylated hydroxytoluene, polyvinyl alcohol,polyoxamers, polyvinyl pyrrollidone, hydroxypropyl methyl cellulose,hydroxyethylmethyl cellulose, and mixtures thereof and mixtures thereof.In some embodiments, the pharmaceutical formulations of this inventionwill not include a preservative.

Where the modulator, e.g. gap junction, connexin, and/or pannexin orpannexin channel modulator is a nucleic acid, such as a polynucleotide,uptake of nucleic acids by mammalian cells is enhanced by several knowntransfection techniques for example those including the use oftransfection agents. Such techniques may be used with certainanti-connexin agents, including polynucleotides. The formulation that isadministered may contain such transfection agents. Examples of theseagents include cationic agents (for example calcium phosphate andDEAE-dextran) and lipofectants (for example Lipofectam™ andTransfectam™), and surfactants.

Where the gap junction, connexin, and/or pannexin modulator comprises apolynucleotide, conveniently, the formulation further includes an agentto assist with cell penetration or the formulation may contain anysuitable loading agent, such as a surfactant, a signalling molecule suchas an antenna peptide, or any other agent suitable for administration tothe eye.

In one embodiment of the invention, the modulator, e.g. gap junction,connexin, and/or pannexin or pannexin channel modulators of the presentinvention are topically administered. Topical formulations of the gapjunction, connexin, and/or pannexin modulators can comprise ointments,gels, which may be, for example, thermosetting gels, drops, sprays,liquids and powders, or a sustained or non-sustained release dosageform.

In some embodiments, the modulator, e.g. the gap junction, connexin,and/or pannexin or pannexin channel modulators, for example, a connexin43 modulator, can be administered as a pharmaceutical compositioncomprising one or a plurality of particles. In some aspects thepharmaceutical composition may be, for example, an immediate releaseformulation or a controlled release formulation, for example, a delayedrelease particle.

In some aspects, a modulator, e.g. pannexin or connexin modulators, forexample, connexin 43 and pannexin 1 modulators, gap junction modulators,etc., can be formulated in a particulate formulation one or a pluralityof particles for selective delivery to the physiological region to betreated. In some embodiments, the particle can be, for example, ananoparticle, a nanosphere, a nanocapsule, a liposme, a polymericmicelle, or a dendrimer. In some embodiments, the particle can be amicroparticle. The nanoparticle or microparticle can comprise abiodegradable polymer.

In some aspects, a modulator, e.g. pannexin or connexin modulators, forexample, connexin 43 and pannexin 1 modulators, gap junction modulators,etc., are formulated to provide compartmentalized release to the site ofadministration. In some aspects of this invention, the modulator may beformulated for compartmentalized release of the modulator, for example,by adjusting the size or coating of the particles. For example, in someaspects, particle formulations of a connexin 43 modulator or a pannexin1 modulator or a hemichannel modulator or a gap junction modulator canbe administered for use in the methods of this invention. Ocular drugdelivery systems comprising particles may comprise, in some aspects,nanoparticles a mean diameter of less than 1,000 nm or microparticlesbetween 1 to 1,000 μm, as determined, for example, by scanning electronmicroscopy. The nanoparticles or microparticles may be, for example,nanospheres or microspheres, or encapsulated nanocapsules andmicrocapsules, in which the connexin modulator is encapsulated in apolymeric or lipid coating. Other methods may be employed to measure theparticle size, including but not limited to light scattering, zetapotential analysis, coulter counting (electrical sensing zone method),and optical microscopy.

The nanoparticle or microparticle can comprise poly(lactic-co-glycolicacid) (“PLGA”) loaded with the gap junction, connexin, and/or pannexinmodulators, for example, connexin 43 modulators. The modulators can beloaded into the particle volume, onto the particle exterior surface, orboth.

In some embodiments, the biodegradable particle can be a particleselected from one of the following particle types: polylactide (PLA)nanoparticles, poly-DL-lactic acid (PDLLA) microspheres or nanospheres,poly (lactic acid) nanoparticles or microparticles, chitosan-modifiedpoly (D,L-lactide-co-glycolide) nanospheres and microspheres (CS-PLGANSs), chitosan-alginate coated nanoparticles or microparticles, solidlipid nanoparticles or microparticles (SLNs), silicon nanoparticles ormicroparticles, polylactic-co-glycolic acid (PLGA) nanoparticles ormicroparticles, polylactic-co-glycolic acid (PLGA) nanoparticles ormicroparticles, pH-sensitive Eudragit P-4135F nanoparticles ormicroparticles, thioketal nanoparticles or microparticles (TKNs) madefrom the polymer poly-PPADT (1, 4-phenyleneacetone dimethylenethioketal), lipopolysaccharides (LPS), alginate nanoparticles ormicroparticles, phospholipid nanoparticles or microparticles, and type Bgelatin enclosed in poly(e-caprolactone) (PCL) microspheres andnanospheres. In some embodiments, the particle is apolylactic-co-glycolic acid (PLGA) nanoparticles or microparticles. Theconnexin 43 modulator may also be entrapped in microcapsules ornanocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,polymeric micelles, liposomes and pegylated liposomes, albuminmicrospheres, microemulsions, nano-particles, and nanocapsules), or inmacroemulsions. Such techniques are disclosed in Remington'sPharmaceutical Sciences (1980) 16th edition, Osol, A. Ed., and hereinincorporated by reference.

In some aspects, the formulation for targeted delivery to the choroidand/or retina can comprise the administering of a modulator, e.g. a gapjunction and/or connexin modulator, or pannexin modulator infusedparticles that present a targeting moiety on the surface of theparticles. In some aspects, the targeting moiety on the surface can becovalently-linked by EDAC coupling(N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride)transferrin protein. In some aspects, the targeting moiety on thesurface can be covalently-linked RGD peptide (with the peptide sequenceGRGDSPK, SEQ ID NO: 233). The particles can be nanoparticles ormicroparticles. In some aspects, the nanoparticles can be from about 200nm to 450 nm in diameter. In some aspects, the particles can comprise apolymer as described above. In some aspects, the particles can comprisepoly-(lactide-co-glycolide). In some embodiments, the gap-junctionand/or connexin modulator can be infused into the particle as theparticle is being synthesized via emulsion preparation. In someembodiments, the gap-junction and/or connexin or pannexin modulator canbe infused into the particles as described in Singh, S. R. et al., GeneTherapy, 16:645-659 (2009) and herein incorporated by reference) afterthe particles have been synthesized by diffusion into the particles.

In some embodiments, the particles can comprise both a peptide fortargeted delivery to the choroid and/or retina for targeted delivery anda peptide for use as a cellular internalization transporter. Theparticle can be a microparticle or nanoparticle, as described herein.The peptide for targeted delivery to the choroid and/or retina can bethe RGD peptide covalently liked to the particle, as described herein.The peptide for use as a cellular internalization transporter can bepresented at the exterior surface of the particle or within the particleas fused to the gap junction and/or connexin modulator. The fusion canbe via a covalent bond, hydrogen bond or bonds, electrostaticinteraction, or van der Walls interaction. The peptide for use as acellular internalization transporter can be any peptide listed in Table65 of this disclosure.

In some embodiments, the modulator, e.g. gap junction and/or connexinmodulator, pannexin modulator or pannexin channel modulator formulatedfor targeted delivery to the choroid and/or retina following peripheraladministration, can be administered by intravenous or intraperitonealinjection. In some aspects, the connexin modulator may be, for example,a connexin 43 modulator. In some aspects, the gap junction and/orconnexin modulator or pannexin modulator formulated for targeteddelivery to the choroid and/or retina following peripheraladministration, can be administered by periocular delivery to the eye.In some aspects, the formulation for periocular delivery can be in theform of eye drops or a gel or a contact lens infused with the gapjunction and/or connexin modulator or pannexin modulator formulation.

In some embodiments, the modulator, e.g. gap junction and/or connexinmodulator, pannexin modulator or pannexin modulator can be formulatedfor targeted delivery by forming a complex with a choroid orretina-targeting agent. The complex can be formed by a covalent bond,hydrogen bond or bonds, electrostatic interaction, or van der Wallsinteraction.

In some aspects, the modulator, e.g. gap junction, connexin, or pannexinorpannexin channel modulators of this invention can be directly bound tocompounds that target the choroid and or retina, or which function as ancell interanalization transporter.

Administering a modulator, e.g. a connexin modulator, for example, aconnexin 43 modulator to the eye of a subject means administering themodulator to the subject, to provide therapeutically effective amountsof the connexin 43 modulator to the eye or specific compartment of theeye. In some instances the connexin modulator, for example, a connexin43 modulator, may be administered by topical, corneal, intravitreal,subconjunctival, or periocular administration. In some aspects,administration may also be intraperitoneal administration. In someembodiments, a microneedle, needle, or implant may be used foradministration of the connexin 43 modulator. The connexin 43 modulatorsof this invention may also be administered to the ciliary body. In someaspects, the connexin 43 modulators of this invention may beadministered via intraventricular, and/or intrathecal, and/orextradural, and/or subdural, and/or epidural routes.

In some aspects, the modulator, e.g. gap junction, connexin 43, pannexin1 and pannexin channel modulators of this invention are formulated forocular administration. The modulators, e.g. connexin 43 and othermodulators may, in some aspects, be formulated to provide controlledand/or compartmentalized release following administration. In someaspects, the formulations may be immediate, or extended or sustainedrelease dosage forms. In some aspects, the dosage forms may compriseboth an immediate release dosage form, in combination with an extendedand/or sustained release dosage form. In some aspects both immediate andsustained and/or extended release of modulators, e.g. connexin 43modulators, can be obtained by combining a modified or unmodifiedconnexin 43 antisense oligonucleotide or polynucleotide, together with amodified or unmodified peptide or peptidomimetic in an immediate releaseform. In some aspects, the dosage forms may be implantable.

In some embodiments, the compositions are isotonic with the fluids ofthe eye, and may have an osmolality of at least about 200 mOsmol/kg,preferably in the range of about 200 to about 350, or about 400mOsmol/kg. The compositions may comprise, for example, sodium chloride,potassium chloride, calcium chloride and/or magnesium chloride.

In some embodiments, any of the modulators, e.g. gap junction channel,pannexin channel, pannexin or connexin modulators of this disclosure,such as connexin 43 and pannexin 1 channel modulators, is formulated toprovide compartmentalized release to the site of administration. In someaspects of this invention, the pannexin or connexin modulator may beformulated for compartmentalized release of the modulator, for example,by adjusting the size or coating of the particles. For example, in someaspects, particle formulations of the modulator, e.g. pannexin modulatoror the connexin modulator, for example, a connexin 43 modulator, can beadministered for use in the methods of this invention. Ocular drugdelivery systems comprising particles may comprise, in some aspects,nanoparticles having a mean diameter of less than 1,000 nm, for example,1-1000 nm, and/or microparticles having a mean diameter between 1 to1,000 μm. The nanoparticles or microparticles may be, for example,nanospheres or microspheres, or encapsulated nanocapsules andmicrocapsules, in which the modulator, e.g. connexin modulator isencapsulated in a polymeric and/or lipid coating.

In some embodiments the formulated modulator is a connexin 43 orconnexin 45 modulator, preferably a connexin 43 modulator.

In other embodiments of any formulation, dose or delivery methoddescribed herein, the modulator is a pannexin channel modulator,preferably a pannexin 1 channel modulator. In still other embodiments ofany formulation, dose or delivery method described herein, the modulatoris a gap junction or hemichannel channel modulator, and preferably insome embodiments, a connexin43 or connexin43 gap junction channel orhemichannel modulator

Particle formulations may be administered, for example, intraocularly byinjection, or intravitreally, subconjunctivally, or periocularly. Thesize and polymer composition of the particles can be selected to controlthe release of the modulator, e.g. connexin 43 modulator from theparticles, for example, the timing and location of release of themodulator. For example, in some aspects of this invention, modulator,e.g. connexin 43 hemichannel or pannexin 1 channel modulators formulatedas poly-lactic acid (PLA) microspheres may be remain in the compartmentof the eye to which they were administered for more than 1, 2, 3, 4, 5,6, or 7 days, or more than one week, more than 10 days, more than 2, 3,4, 5, or 6 weeks, or longer. In some aspects, modulator microparticlesdelivered intravitreally by injection or a microneedle may providesustained release of the modulator. In other aspects, smaller particlessuch as nanoparticles comprising modulators, may diffuse rapidly fromone compartment of the eye to another, any may be readily internalizedin ocular tissues and cells of the anterior and posterior segments ofthe eye. Intravitreal delivery of polylactide nanoparticles comprisingconnexin 43 or other modulators may, in some aspects of this invention,provide delivery across the retina with preferential localization inretinal pigment epithelium cells for 1, 2, 3, 4, 5, 6, or 7 days, ormore than one week, more than 10 days, more than 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, or more than 16 weeks. Nanosphere andmicrosphere ocular drug delivery formulations of connexin 43 or othermodulators can, in some aspects, enhance cellular penetration, improvebioavailability, for example, by protecting against degradation, and/orprovide sustained delivery. In some embodiments, formulations comprisinglarger nanoparticles (greater than 150, 200, 300, 400, 500, 600, 700,800, 900 nm) or microparticles, for example, particles having a meandiameter between 150 nm and 9 micrometers, or between 1 and 9micrometers, for example, 1, 2, 3, 4, 5, 6, 7, 8 micrometers, or anyrange between any two of the recited mean diameters may be suitable forcompartmentalized ocular administration as a result of slower releaseand/or diffusion from the particles of this size.

The particle formulations of any of the pannexin, or connexin modulatorsof this disclosure, such as connexin 43 modulators may also compriseliposomes. In some aspects, the liposomes may have a diameter rangingbetween 50 nm to a few micrometers. In some aspects the liposomes can beinjected, or applied topically or subconjunctivally to a segment orcompartment of the eye, and may, in some embodiments, provide slow drugrelease from a relatively inert dosage form. The liposome formulationsmay also have fewer side effects, because only a limited amount ofconnexin 43 agents or other ocular treatment compounds come in directcontact with ocular tissues.

Increased corneal penetration into the anterior segments can be achievedwith the addition of permeability enhancers to the drug formulation.Cell penetration agents may also be used to enhance deliver of theconnexin 43 modulator to RGC neurons or other cells in the eye.Formulations may also contain, for example, surfactants, bile acids,chelating agents, and/or preservatives. In some embodiments,formulations for intravitreal administration may be free ofpreservatives.

As used herein, “matrix” includes for example, matrices such aspolymeric matrices, biodegradable or non-biodegradable matrices, andother carriers useful for making implants or applied structures fordelivering the connexin modulators and/or ocular neuropathic treatmentagents.

Ocular Implants

In some embodiments, the dosage forms of this invention may be ocularimplants. In some embodiments, the implants may be implanted, forexample intravitreally, suprachoroidally, intrasclerally, orsubconjunctivally. Ocular implants deliver sustained levels ofmodulator, e.g. connexin and/or pannexin modulators, to the desiredocular site, and bypassing the blood-brain barrier. Implants may beimplanted, for example, subconjunctivally, into the episcleral orintrascleral space, in contact with the sclera, into the vitrous cavity.Intrascleral administration for use with the connexin modulators of thisinvention may, in some embodiments, be useful for delivery to theposterior segment of the eye, with lower systemic absorption of themodulator than subconjunctival or peribulbar administration. Implantsmay also be placed intravitreally, which permits delivery to theposterior segment of the eye. In some embodiments, the implant can beinserted intravitreally through a sclerotomy site, for example, over thepars plana.

Subconjunctival implants may be inserted through a small incision in theconjunctiva and placed in contact with the sclera. Intrascleral devices,implanted in a small scleral pocket at one-half the total scleralthickness, may also be used, and are useful for delivery to theposterior of the eye with less systemic absorption of the drug thansubconjunctival or peribulbar injections.

Intravitreal placement of ocular implants also permits delivering a drugdirectly to target tissues of the posterior segment. The implant may beinserted into the vitreous, for example, through a sclerotomy site, orinjected with an applicator. The site of implantation is commonly overthe pars plana, which is anterior to the insertion of the retina andposterior to the lens, to minimize damage to those structures.

In some embodiments where the dosage form is an implant, the implant,may, in some embodiments, comprise at least one copolymer coatingcomprising a connexin 43 modulator and a biodegradable and biocompatiblecopolymer and at least one coating comprising a biodegradable polyesterand a connexin 43 modulator. In some embodiments, the dosage form mayhave an inner coating comprising a biodegradable polyester and aconnexin 43 modulator and an outer coating comprising a connexin 43modulator and a biodegradable and biocompatible copolymer.

The dosage form core or implant may comprise a connective tissue blendedwith a biodegradable polymer. In some embodiments, the connective tissuemay comprise one or more of the following connective tissues: collagen,elastin, and chondroitin-4-sulfate. In some embodiments, the connectivetissue may be present at an amount about 50-99% collagen (w/w). In someembodiments, the biodegradable polymer may comprise a biodegradablepolyester polymer. In some embodiments, the polyester polymer maycomprise one or more of the following selected biodegradable polyesterpolymers: poly(L-lactide), poly(glycolide), poly(DL-lactide),poly(dioxanone), poly(DL-lactide-co-L-lactide),poly(DL-lactide-co-glycolide), poly(glycolide-co-trimethylenecarbonate), and poly(caprolactone) (“polycaprolactone”). In someembodiments, the polyester polymer may comprise polycaprolactone (PCL).In some embodiments, the amount of polycaprolactone may be present at anamount about 1-50% polycaprolactone (w/w). In some embodiments, themolecular weight of the polycaprolactone may range from 10,000 Da to3,000,000 Da.

In some embodiments, the collagen and the polymer comprising the implantmay be electrospun into fibers to create the implant or core, or ascaffold sheet, from which the desired implant form may be obtained.Alternatively, by controlling fiber orientation during deposition, athree-dimensional implant may be fabricated having a desired shape.Three-dimensional printing may also be used to obtain a core forimplantable dosage forms of this invention. Alternatively the scaffoldsubstrate may be extracted from a natural source and reonstitute itselfupon processing into a suitable matrix such as collagenous materialsextracted from marine organisms, including but not limited to jellyfishor from mammalian sources including but limited to bovine, equine, ovinesources.

In some embodiments, the implant may be coated with a mixture comprisinga modulator, e.g. a connexin or pannexin channel modulator, for example,a connexin 43 or hemichannel modulator. The mixture may also comprise apolymer that temporarily binds the modulator, e.g. pannexin or connexinchannel modulator, to the implant (“binding polymer”, or “elutingpolymer”). Such polymers may include, but are not limited to, one ormore of the following homopolymers or copolymers of the followingselected polymers: poly(lactic-co-glycolic acid (PLGA), poly(L-lactide),poly(glycolide), poly(DL-lactide), poly(dioxanone),poly(DL-lactide-co-L-lactide), poly(DL-lactide-co-glycolide),poly(glycolide-co-trimethylene carbonate), and polycaprolactone (PCL),Multiple coatings may be applied to the implant to achieve a desiredelution profile of the connexin 43 modulator. In some embodiments, eachcoating layer may be applied by immersing the core or implantable formin a solution of the dissolved binding polymer and the modulator, e.g.connexin or pannexin channel modulator, for example, a connexin 43hemichannel or gap junction modulator. The core or implantable form maythen be removed and freeze-dried (lyophilized) to remove the solvent.Alternatively, the core or implantable form may be removed and subjectto quick evaporation by placing the core or implantable form in a vacuumchamber. The core or implantable form comprising the first bindingpolymer layer may be subsequently immersed into another solutioncomprising the same or another connexin or pannexin modulator, forexample, a modulator, e.g. connexin 43 and/or pannexin 1 channelmodulator, than that used in a prior immersion, and further comprisingthe same binding polymer as used in the prior immersion, or a differentbinding polymer. The steps of immersion and lyophilization may berepeated up to twenty times to create one or more layers of coatingand/or one or more coatings on the core or implantable form.

Practitioners in the art will appreciate that the polymer whichtemporarily binds the anti-connexin agent to the core or implantableform may comprise any polymer suitable for drug-elution in implants.Such polymers exhibit the following properties: are non-toxic(proportional to their beneficial effect), are metabolized directly orwhose hydrolysis products are metabolized, and may easily be sterilized.A review of such applicable materials is found in J. C. Middleton, A. J.Tipton, Biomaterials, 21 (2000), 2335-2346, herein incorporated byreference.

Nonbiodegradable or biodegradable polymeric devices for controlled orsustained release may also be used in the methods of this invention.Nonbiodegradable implants can provide steady, controlled release of adesired modulator(s). Biodegradable implants can be manufactured into adesired shape, can be injected in an office or outpatient procedure, anddo not require removal.

Nonbiodegradable (Reservoir) Implants

Reservoir implants are typically made with a pelleted drug coresurrounded by nonreactive substances such as silicon, ethylene vinylacetate (EVA), or polyvinyl alcohol (PVA); these implants arenonbiodegradable and can deliver continuous amounts of a drug for monthsto years.

In some embodiments, the modulators of this invention may be formulatedas implantable nonbiodegradable reservoir dosage forms suitable fordelivering the connexin modulators, alone, or in combination with otherocular neuropathic treatment agents, for a sustained period of time, forexample, over 1 month, or over 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, or over 36 months. Insome embodiments, the reservoir dosage forms may comprise a corecomprising one or more modulators as described herein, alone, or incombination with other ocular neuropathic treatment agents as describedherein, surrounded by a nonreactive coating comprising silicon, such aspolydimethylsiloxane (PDMS), ethylene vinyl acetate (EVA), or polyvinaylalcohol (PVA).

Biodegradable Matrix Implants

The dosage forms of this invention may also comprise copolymer implantsuseful in delivering a loading dose of the modulator, e.g. connexinand/or pannexin channel modulator, as well as tapering doses of themodulator over a sustained period of time lasting up to 6-months ormore. Copolymer implants may be made from the copolymerspoly-lactic-acid (PLA) and/or poly-lactic-glycolic acid (PLGA), whichare biodegraded to water and carbon dioxide. The rate and extent ofrelease of the modulator from the implant can be decreased by alteringthe relative concentrations of lactide (slower release) and glycolide(faster release), altering the polymer weight ratios, adding additionalcoats of polymer. Unlike Nonbiodegradable implants, biodegradableimplants do not require removal, and provide flexibility in dose andtreatment from a short duration of weeks to more sustained release ofmonths to a year or more, depending on the polymer PLA/PLGA ratio. Insome aspects, biodegradable dosage forms can be personalized inaccordance with the subject's disease progression. In some aspects, thebiodegradable matrix implant can be, for example, a dissolvable diskmaterial such as that described in S. Pflugfelder et al., ACS Nano, 9(2), pp 1749-1758 (2015).

Microneedles

In some embodiments, the modulator compounds and formulations of thisinvention can be administered via microneedles. Microneedles areindividual needles or arrays of micrometer-sized needles, as describedin Kim, Y. et al., Invest. Ophthalmol. Vis. Sci. Nov. 13, 2014 vol. 55no. 11 7376-7386. In some embodiments, microneedles can be from 500 to750 micrometers in length, and can be coated with the formulationsdescribed herein. An array of microneedles can be used for theadministration of formulations of this invention. The shafts of solidmicroneedles can be coated with formulations of this invention whichthen dissolve into the regions of the eye after insertion.

In some embodiments, a modulator, e.g. a connexin 43 and/or a pannexin 1channel modulator, for example, is administered to the subject,providing therapeutically effective amounts of the connexin 43 modulatorto the eye or specific compartment of the eye. In some instances themodulator(s), may be administered by topical, intravitreal,subconjunctival, or periocular sub-tenon administration. In someaspects, administration may also be intraperitoneal or other form ofsystemic administration. In some embodiments, a microneedle, microneedlearray, needle, or implant may be used for administration of themodulator(s). In some embodiments a microneedle may be used toadminister a modulator as described herein, for example, a connexin 43modulator, a pannexin channel modulator, a gap junction modulator,and/or a pannexin modulator to the eye, or a specific compartment orstructure of the eye, for example, the choroid, suprachoroidal space, orretina, or sclera. For example, a hollow microneedle may be insertedinto the sclera, suprachoridal space and the modulator may beadministered and/or infused into that location or compartment of theeye. Administration to the choroid may, in some embodiments, beperformed by administering the modulator to the suprachoroidal space,from which the infused drug flows circumferentially within thesuprachoroid space toward the choroid, retina, macula, optic nerve andother structures and compartments in the back of the eye. In someembodiments, the microneedle may be inserted through the sclera into thesuprachoroidal space without penetrating the choroid. In someembodiments, the microneedle may be retracted following infusion. Insome embodiments the penetration of the microneedle may be controlled toa desired depth within a tissue, stroma, and/or compartment of the eye.In some embodiments the microneedle may also be coated with themodulators of this invention or other ocular drug agents. In someaspects the volume of modulator and/or ocular drug agent administered bymicroneedle may be from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125,130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195,200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265,270, 275, 280, 295, or 300 μl, or any range of volume between any two ofthe recited numbers or any volume between any two recited numbers. Anysuitable formulation of this invention may be administered bymicroneedle injection, including, for example, nanoparticle ormicroparticle formulations, or other formulations injectable bymicroneedle.

In some aspects, the microneedle comprises a base from which one or moremicroneedles extend, typically in a direction perpendicular to the base.The microneedle may be solid or hollow. A hollow microneedle maycomprise, for example, a single, straight bore through the center of themicroneedle, or multiple bores. In some embodiments the hollowmicroneedles may comprise bores that follow complex paths through themicroneedle, multiple entry and exit points from the bore(s), andintersecting or networks of bores that form one or more continuouspathways from the base of the microneedle to an exit point in the shaftand/or tip of the microneedle. See, e.g., U.S. Pat. Nos. 7,918,814,8,197,435, 8,636,713 and 8,808,225. In some embodiments, microneedlescan be from 50 to 2000 micrometers in length, from 50 to 500 micrometersin width (diameter), and can be coated with the formulations describedherein. The microneedle can comprise a shaft and a tip. The shaft andtip can be in contact. The tip of the microneedles can be straight ortapered. The tapered microneedle tip can be a point, or a blunt end. Insome embodiments, drilling microneedles may also be used. See, forexample, U.S. Publication 20050137525. Other microneedles are describedin, for example, U.S. Patent Publication Nos. 2006/0086689,2006/0084942, 2005/0209565, 2002/0082543, U.S. Pat. Nos. 6,334,856,6,611,707, and 6,743,211.

The microneedle and/or ocular implants can comprise one or more suitablebiocompatible materials, including metals, glasses, semi-conductormaterials, ceramics, or polymers, for example, pharmaceutical gradestainless steel, gold, titanium, nickel, iron, gold, tin, chromium,copper, and alloys thereof. Polymers for use in microneedles and/orocular implants can be biodegradable or non-biodegradable. Examples ofsuitable biocompatible, biodegradable polymers includepoly(lactic-co-glycolic acid (PLGA), poly(L-lactide), poly(glycolide),poly(DL-lactide), poly(dioxanone), poly(DL-lactide-co-L-lactide),poly(DL-lactide-co-glycolide), poly(glycolide-co-trimethylenecarbonate), and polycaprolactone (PCL), polyanhydrides, polyorthoesters,polyetheresters, polyesteramides, poly(butyric acid), poly(valericacid), polyacrylamides, polyacrylates, polyurethanes and copolymers andblends and crosslinked variants thereof. The microneedles can compriseone or more shafts for the administration of formulations of thisinvention. The shafts can be porous or non-porous. The shafts can behollow or solid. The microneedles can further comprise a tip that iscomposed of a material which is different from the shaft. The tip of themicroneedles can be of a different hardness (as measured by durometry)than the shaft of the microneedles. The tip or shaft or both of themicroneedles can be biodegradable or non-biodegradable. Thenon-biodegradable microneedle tip or shaft can be composed of metals,glasses, semi-conductor materials, ceramics, or polymers. Examples ofsuitable metals include pharmaceutical grade stainless steel, titanium,gold, silver, nickel, iron, gold, tin, chromium, copper, and alloysthereof. Representative non-biodegradable polymers include variousthermoplastics or other polymeric structural materials known in thefabrication of FDA-approved medical devices. Examples include nylons,polyesters, polycarbonates, polyacrylates, polymers of ethylene-vinylacetates and other acyl substituted cellulose acetates, non-degradablepolyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride,poly(vinyl imidazole), chlorosulphonate polyolefins, polyethylene oxide,blends and copolymers and crosslinked variants thereof. Thebiodegradable microneedle tip or shaft can be formulated to dissolveafter administration of the formulations described herein, such that anyresidual microneedle material which may be present at the administrationsite can be dissolved in the body of the subject. For example, themicroneedle can be composed of a material which is formulated to be asolid material (optionally, as a function of temperature) duringinjection, and which after injection (and optionally, exposure to thesubject's temperature) partially or fully degrades. Such microneedlescan optionally be composed of water-soluble polymers, water-solubleinorganic materials, or ice. Biodegradable microneedles can provide anincreased level of safety compared to non-biodegradable ones, such thatthey are essentially harmless even if inadvertently broken off into theocular tissue.

Microneedles can be inserted into any compartment or structure of theeye to administer the moculator compounds and formulations describedherein. Microneedles can be inserted into one or more of the choroid,the retina, the sclera, the suprachoroidal space, the Bruch's membrane,the retinal pigment epithelium, the subretinal space, the macula, theoptic disk, the optic nerve, the ciliary body, the trabecular meshwork,the aqueous humor, or the vitreous humor for the administration of theformulations described herein. For example, a microneedle can beinserted perpendicularly into the sclera, reaching the suprachoroidalspace in a short penetration distance. The delivery of the formulationsdescribed herein into the suprachoroidal region allows for the deliveryof the formulation over a larg tissue area and affords targetingdifficult to target tissues in a single administration as comparedsingle canula needle administration. In one embodiment, theadministration can be confined to one specific structure or compartmentof the eye. The specific structure or compartment of the eye to whichthe microneedle administration is localized can be one of the following:the choroid, the retina, the sclera, the suprachoroidal space, theBruch's membrane, the retinal pigment epithelium, the subretinal space,the macula, the optic disk, the optic nerve, the ciliary body, thetrabecular meshwork, the aqueous humor, or the vitreous humor. Notwishing to be bound by any theory, it is believed that upon entering thesuprachoroidal space the administered formulation flowscircumferentially from the insertion site toward the retinochoroidaltissue and optic nerve in the posterior segment of the eye as well asanteriorly toward the uvea and ciliary body. Furthermore, a portion ofthe administered formulation may remain in the sclera near themicroneedle insertion site, serving as additional reservoir of theadministered formulation that subsequently can diffuse into thesuprachoroidal space and other adjacent tissues. As defined herein,“suprachoroidal space,” also known as suprachoroid or suprachoroidia,describes the potential space in the region of the eye disposed betweenthe sclera and choroid.

The administration of the formulations described herein usingmicroneedles can afford an extended duration of the modulator, e.g.connexin or pannexin channel modulating agent within the targeted regionor structure of the eye. The microneedle delivery method affords alonger duration of the administered formulation than if the formulationwere administered via topical application of the formulation to theocular tissue.

Administering a modulator and/or ocular drug agent of this invention totissue or a compartment of the eye the eye with a microneedle maycomprise inserting a microneedle into the ocular tissue and depositing adrug formulation into the ocular tissue. In some embodiments, at leastone microneedle is inserted into the tissue of the eye withoutpenetrating across the tissue stroma. In one embodiment, the method ofadministration may further include partially retracting the hollowmicroneedle after the insertion step and before and/or during thedepositing of the drug formulation, which, in some embodiments, may forma space for the infusion of the modulator and/or ocular drug agent. Insome embodiments the modulator is, for example, a connexin modulator, agap junction modulator, or a pannexin or pannexin channel modulator.

The administration via a microneedle can be controlled by controllingthe infusion pressure. The infusion pressure will concomitantlydetermine the amount of drug being delivered. In some embodiments, theinfusion pressure may be at least 125 kPa, at least 150 kPa, at least175 kPa, at least 200 kPa, at least 250 kPa, or at least 300 kPa. Theinfusion pressure through the microneedle can be generated by diffusion,capillary action, a mechanical pump, electroosmosis, electrophoretic,convection or other driving forces. The infusion pressure can optionallybe monitored by measuring the backpressure so as to regulate thecontinuous pressure. The infusion pressure can optionally be controlledby microcontrollers, sensors, mechanical force-feedback circuits,actuators, valves, or pumps.

Combinations of Connexin or Pannexin Modulators with Rho KinaseInhibitors or Other Agents

Open-angle glaucoma is characterized by abnormally high resistance tofluid (aqueous humor) drainage from the eye. Normal resistance isrequired to provide an intraocular pressure sufficient to maintain theshape of the eye for optical integrity. The resistance is provided bythe trabecular meshwork, a complex, multilaminar tissue consisting ofspecific cells with a dense actomyosin cytoskeleton network, collagenousbeams and extracellular matrix. In the glaucomatous eye, the rate ofaqueous humor production remains constant, while the increasedresistance to outflow is responsible for the elevated intraocularpressure. Rho-kinase functions as a key downstream mediator of Rho andis ubiquitously expressed. Rho-kinase enzymes are serine/threoninekinases that regulate the function of a number of substrates includingcytoskeletal proteins such as adducing, moesin, sodium ion-proton ionexchanger 1 (NHE1), LIM-kinase and vimentin, contractile proteins suchas the myosin light chain phosphatase binding subunit (MYPT-I), CPI-17,myosin light chain and calponin, microtubule associated proteins such asTau and MAP-2, neuronal growth cone associate proteins such as CRMP-2,and transcription factors such as serum response factor (Loirand et al.,Circ. Res. 98:322-334 (2006)). Rho-kinase is also required for cellulartransformation induced by RhoA. Rho-kinase is an intermediary ofmultiple signaling pathways, regulating a variety of cellular phenomenaincluding cytoskeletal rearrangement, actin stress fiber formation,proliferation, chemotaxis, cytokinesis, cytokine and chemokinesecretion, endothelial or epithelial cell junction integrity, apoptosis,transcriptional activation and smooth muscle contraction. As a result ofthese cellular actions, rho-kinase enzymes regulate physiologicprocesses such as vasoconstriction, bronchoconstriction, tissueremodeling, inflammation, edema, platelet aggregation and proliferativedisorders. Rho kinase acts on the trabecular meshwork to maintain theTOP. Accordingly, inhibiting rho kinase will increase fluid outflowthrough the trabecular meshwork thus decreasing the TOP via a mechanismwhich is different from connexin and/or pannexin or hemichannel and/orpannexin channel modulation to reduce TOP.

In some embodiments, Rho kinase inhibitors are used to treat normalpressure glaucoma.

Inhibitors of rho kinase have been shown to reduce TOP in mammals byincreasing aqueous humor drainage through the trabecular meshwork (Tianand Kaufman, Arch Ophthalmol. 122: 1171-1178, 2004). The inventorsappreciate that the coadministration of a rho kinase inhibitor with amodulator, e.g. connexin or pannexin channel modulator will work on twoindependent mechanisms for reducing TOP in a subject, thereby resultingin a more efficacious treatment than any single treatment.

Additional agents for coadministration with the modulator, e.g. connexinor pannexin channel modulators can include other treatments whichotherwise reduce TOP, including but not limited to lantanoprost,anti-VEGF compounds, Timolol, Brimonidine, Brimonidine tartarate,Rescula (unoprostone isopropyl ophthalmic solution, 0.15%), Dorzolamide,Roclatan, and AR-13533 (Aerie Pharmaceuticals, Bedminster, N.J.). Byacting on mechanisms other than connexin or pannexin channel modulation,the coadministration of multiple treatments for treating oculardisorders can result in synergistic effects such as (1) reduced overalldosages, (2) reduced overall side effects, (3) reduced overall treatmentfrequency, and (4) increased patient compliance and thus efficacy oftreatment.

In some aspects, the VEGF modulators for use in this invention areantagonists that inhibit and/or block VEGF or that inhibit and/or blockupstream agonists of VEGF. In some aspects the VEGF antagonists include,for example, antagonists that bind to and inhibit VEGF, compounds thatinhibit expression of VEGF, and/or viral vectors comprising VEGFinhibitors or encoding proteins or antisense polynucleotides that blockor inhibit VEGF. In some aspects, species that inhibit VEGF and/orupstream agonists of VEGF are, for example, antibodies or antibodyfragments, nanobodies, peptide or peptidomimetics, receptor fragments,recombinant fusion proteins, aptamers, small molecules, or single chainvariable fragments (scFv). In some aspects, VEGF antagonist antibodiesare, for example, Lucentis™ (ranibizumab), and/or Avastin™(bevacizumab).

In some aspects, the VEGF antagonist can be an antibody to VEGF, forexample, Lucentis™ (ranibizumab), or Avastin™ (bevacizumab).

In some aspects, VEGF antagonists which are antisense to upstreamagonists of VEGF species that bind to and therefore inhibit VEGF can bea RTP801 inhibitor or REDD1 blocker. In some aspects, the RTP801inhibitor or REDD1 blocker can be PF-655 (by Quark Pharmaceuticals andPfizer), also known as REDD14NP or RTP801i). In some aspects the REDD1blocker can have the mRNA sequence 5′-AGCUGCAUCAGGUUGGCAC-3′ (SEQ ID NO:234).

In some aspects of this invention, the VEGF antagonist is, for example,a peptide or peptidomimetic, for example, pegaptanib sodium (Macugen™),and AGN-150998. Macugen™ is a modified RNA sequence,((2′-deoxy-2′-fluoro)C-Gm-Gm-A-A-(2′-deoxy-2′-fluoro)U-(2′-deoxy-2′-fluoro)C-Am-Gm-(2′-deoxy-2′-fluoro)U-Gm-Am-Am-(2′-deoxy-2′-fluoro)U-Gm-(2′-deoxy-2′-fluoro)C-(2′-deoxy-2′-fluoro)U-(2′-deoxy-2′-fluoro)U-Am-(2′-deoxy-2′-fluoro)U-Am-(2′-deoxy-2′-fluoro)C-Am-(2′-deoxy-2′-fluoro)U-(2′-deoxy-2′-fluoro)C-(2′-deoxy-2′-fluoro)C-Gm-(3′→3′)-dT)(SEQ ID NO: 235), 5′-ester withα,α′-[4,12-dioxo-6-[[[5-(phosphoonoxy)pentyl]amino]carbonyl]-3,13-dioxa-5,11-diaza-1,15-pentadecanediyl]bis[w-methoxypoly(oxy-1,2-ethanediyl)],sodium salt. AGN-150998/MP0112 is an anti-VEGF DARPin, a small proteinthat binds to VEGF.

In some aspects of this invention, the VEGF antagonist is, for example,a recombinant fusion protein such as, for example, aflibercept (Eyelea™)or conbercept. Aflibercept is a recombinant fusion protein consisting ofportions of human VEGF receptors 1 and 2 extracellular domains fused tothe Fc portion of human IgG1. Conbercept is a recombinant fusion proteincomposed of the second Ig domain of VEGFR1 and the third and fourth Igdomains of VEGFR2 to the constant region (Fc) of human IgG1.

In some aspects, the scFv VEGF antagonist is, for example, ESBA1008.ESBA1008 is a humanized monoclonal single-chain FV (scFv) antibodyfragment targeting VEGFA.

In some aspects, the viral vector VEGF antagonist can be AAV-sFLT01(also known as “AVA-101”). AAV2-sFlt01 is an adeno-associated viralvector that carries the gene construct for a secreted chimericprotein—sFLT01—that binds to VEGF. sFLT01 is a VEGF-binding protein thatconsists of domain 2 of Flt-1 linked to a human immunoglobulin G₁ heavychain Fc fragment (sFlt01), combined with an adeno-associated virus(AAV) to produce AAV2-sFlt01.

In some aspects of this invention, VEGF antagonists are small molecules,for example, Vatalanib, Cediranib, AL39324, Pazopanib, TG100572, orTG100801. Vatalanib(N-(4-chlorophenyl)-4-(pyridin-4-ylmethyl)phthalazin-1-amine) is alsoknown as PTK787, PTK/ZK, or CGP 79787. Cediranib, also known as AZD2171, Recentin™, ZD 2171, or CAS Number 288383-20-0, is also known as4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxy-7-[3-(1-pyrrolidinyl)propoxy]-quinazoline.AL39324, also known as Linifanib, CAS No. 796967-16-3, 1145655-58-8 (asthe HCl salt), or 796967-17-4 (as the trifluoroacetate salt), is alsoknown as1-[4-(3-amino-1H-indazol-4-yl)phenyl]-3-(2-fluoro-5-methylphenyl)urea.Pazopanib, also known as Votrient™, Armala™, or Patorma™, is also knownas5-[[4-[(2,3-dimethyl-2H-indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2-methylbenzenesulfonamidemonohydrochloride. TG100801 is a pro-drug version of TG100572, and isalso known as4-Chloro-3-(5-methyl-3-((4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-amino)benzo[e][1,2,4]triazin-7-yl)p;4-Chloro-3-[5-methyl-3-[[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]amino]-1,2,4-benzotriazin-7-yl]phenol1-benzoate.

In some aspects, mTOR inhibitors for use in the methods of thisinvention are, for example, macrolides or small molecules. In someembodiments, the macrolide mTor inhibitors are, for example,temsirolimus, sirolimus, or everolimus. Temsirolimus, also known asTorisel™, or CCI-779, is also known as(1R,2R,4S)-4-{(2R)-2-[(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetracosahydro-3H-23,27-epoxypyrido[2,1-c][1,4]oxazacyclohentriacontin-3-yl]propyl}-2-methoxycyclohexyl3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate. Sirolimus is also knownas rapamycin, Rapamune™, AY-22989, Perceiva™, WY-090217, or(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]-oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone.Everolimus, also known as Afinitor™, Certican™, Votubia™, or Zortress™,is also known asdihydroxy-12-[(2R)-1-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.0hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone. This inventionalso features small molecule mTor inhibitors such as, for example,Palomid 529, XL388, or Dactolisib. Palomid 529 is also known as3-(4-methoxybenzyloxy)-8-(1-hydroxyethyl)-2-methoxy-6H-benzo[c]chromen-6-one.XL388 is also known as[7-(6-amino-3-pyridinyl)-2,3-dihydro-1,4-benzoxazepin-4(5H)-yl][3-fluoro-2-methyl-4-(methylsulfonyl)phenyl]-methanone.Dactolisib, also known as BEZ235, or NVP-BEZ235, is also known as2-methyl-2-(4-(3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl)phenyl)propanenitrile.

In some aspects, PDGF modulators for use in the methods of thisinvention include, for example, Fovista™ (also known as E-10030), aanti-platelet-derived growth factor (anti-PDGF-B) aptamer.

In some aspects, PEDF modulators for use in the methods of thisinvention include, for example, AdGVPEDF.11D (GenVec). AdGVPEDF.11D is aadenovirus vector containing the gene for PEDF. AdGVPEDF.11D uses anadenovector to deliver the PEDF gene to target cells, resulting in thelocal production of PEDF in the treated eye. Pigment epithelium-derivedfactor (PEDF) is a potent inhibitor of new vessel growth. PEDF is alsoknown as serpin F1.

In some aspects, S1P production blockers for use in the methods of thisinvention include, for example, sonepcizumab, also known as LT1009,Asonep, Sphingomab™, or iSONEP™. iSONEP™ is a humanized monoclonalantibody that binds to sphingosine 1-phosphate (S1P).

In some aspects, the ocular treatment agent for use in combination withone or more of the modulator, e.g. gap junction, connexin and/orpannexin channel modulators, of this invention is, for example,squalamine. Squalamine is also known as Evizon™, or(1S,2S,5S,7R,9R,10R,11S,14R,15R)—N-{3-[(4-aminobutyl)amino]propyl}-9-hydroxy-2,15-dimethyl-14-[(2R,5R)-6-methyl-5-(sulfooxy)heptan-2-yl]tetracyclo[8.7.0.0{circumflexover ( )}{2,7}.0{circumflex over ( )}{11,15}]heptadecan-5-aminium.

In some aspects, tubulin binding agents for use in the methods of thisinvention include, for example, combretastatin, combretastatin A-4phosphate, combrestastin derivatives, or OC-10X. Combretastatin is alsoknown as 2-Methoxy-5-[(Z)-2-(3,4,5-trimethoxy-phenyl)-vinyl]-phenol.OC-10X (OcuCure, Roanoke, Va.) is a low-molecular-weight quinazolinone.The tubulin binding agents can also be beta-lactam based derivatives ofcombrestastin, as described in O'Boyle, N. et al. Journal of MedicinalChemistry 53 (24): 8569-8584, 2010. doi:10.1021/jm101115u. PMID21080725, and herein incorporated by reference.

In some aspects, integrin inhibitors for use in the methods of thisinvention include, for example, ALG-1001, Volociximab, or JNJ-26076713.ALG-1001 (Allegro Ophthalmics) is a small peptide. Volociximab, alsoknown as M200, or Opthotec™, is a chimeric monoclonal antibody thatbinds to and inhibits the functional activity of α5β1 integrin.JNJ-26076713 is a tetrahydroquinoline-containing αVβ3/αVβ5 integrinantagonist and is also known as 3-Quinolinepropanoic acid,1,2,3,4-tetrahydro-beta-((1-(1-oxo-3-(1,5,6,7-tetrahydro-1,8-naphthyridin-2-yl)propyl)-4-piperidinyl)methyl)-,(betaS,3S)—.

Ocular treatment agents for use in combination with one or more of themodulator, e.g. gap junction, connexin, and/or pannexin or pannexinchannel modulators, of this invention also include, for example,complement modulators, and other therapeutic agents useful, for example,in treating geographic atrophy, dry AMD, non-exudative AMD, and/orDrusen development.

Complement modulators for use as ocular treatment agents in combinationwith one or more modulators, e.g. gap junction, connexin, and/orpannexin or pannexin channel modulators, are, for example, compstatin,TP10, Eculizumab, ARC1905, JPE-1375, PMX53, Lampalizumab, or rhCFHp.Compstatin, also known as AL-78898A or POT-4, is a cyclic peptide withthe sequence -[ICVVQDWGHHRCT]- (SEQ ID NO: 22). TP10 (AvantImmunotherapeutics) is a recombinant protein and is an inhibitor ofsoluble complement receptor one (sCR1). Eculizumab, also known asSoliris™, or Solaris™, is a humanized monoclonal antibody. ARC1905, alsoknown as Zimura™ is a PEGylated, stabilized aptamer targeting complementfactor C5. JPE-1375 is a small molecule peptidomimetic antagonisttargeting C5aR, the receptor for complement factor C5a. PMX53 is acyclic hexapeptide, with the sequence Ace-Phe-[Orn-Pro-dCha-Trp-Arg].Lampalizumab, also known as anti-factor D, or FCFD4514S, is a monoclonalantibody that inhibits complement factor D. rhCFHp is a recombinantfusion protein form of full-length Factor H in its protective to correctabnormal Factor H activity.

In some aspects, ocular treatment agents for use in combination with oneor more of modulator, e.g. gap junction, connexin, and/or pannexin orpannexin channel modulators, of this invention include TNF-alphainhibitors, C-raf kinase inhibitors, NSAIDs, or nAChR inhibitors. Insome embodiments, the TNF-alpha inhibitor can be Adalimumab orinfliximab. In some embodiments, the C-raf kinase inhibitor can beiCo-007. In some embodiments, the NSAID can be Brofenac. In someembodiments, the nAChR inhibitor can be mecamylamine.

Ocular treatment agents for use in combination with one or more of themodulator, e.g. gap junction, connexin, and/or pannexin or pannexinchannel modulators, of this invention can also include pharmaceuticalagents such as verteporfin (e.g., Chlorin™, Visudyne, talaporfin sodium(e.g., Aptocine™, Laserphyrin™, Litx™), isopropyl unoprostone (e.g.,Ocuseva™, Rescula™), interferon beta (e.g., Feron™), fluocinoloneacetonide (e.g., Envision TD™, Retisert™), dexamethasone (e.g.,Osurdex™, Ozurdex™, Posurdex™, Surodex™), canakinumab (e.g., Ilaris™),bromfenac (Bromday™), ophthalmic (e.g., Bronac™, Bronuck™, Xibrom™,Yellox™), brimonidine (e.g., Alphagan™, Bromoxidine™, Enidin™),anecortave acetate (e.g., Retaane™, Edex™, Prostavasin™, Rigidur™,Vasoprost™, Viridal™), VEGF-Trap-Eye™, ocriplasmin (e.g., Iluvien™,Medidur™, Medidur FA™), NT-501, KH-902, fosbretabulin tromethamine(e.g., Zybrestat™), AL-8309, aganirsen (e.g., Norvess™), volociximab(e.g., Opthotec™), triamcinolone (e.g., Icon Bioscience), TRC-105,Burixafor (e.g., TG-0054), TB-403 (e.g., R-7334), SB-623, S-646240,RTP-801Ï-14 (e.g., PF-4523655), RG-7417 (e.g., FCFD-4514S), PG-11047(e.g., CGC-11047), padeliporfin (e.g., Stakel™), OT-551, ontecizumab,NOX-A12, hCNS-SC, Neu-2000, NAFB001, MA09-hRPE, LFG-316, iCo-007 (e.g.,ISIS-13650), hl-conl, GSK-933776A, GS-6624 (e.g., AB-0024), epitalon,dalantercept, MP-0112, CNTO-2476, CERE-120, CCX-168, Brimonidine-DDS,bevasiranib sodium (e.g., Cand5), bertilimumab, ACU-4429, A6 (e.g.,Paralit™), TT-30, sFLT-01 gene therapy, RetinoStat™, PRS-050 (e.g.,Angiocal™), PF-4382923, MC-1101, GW-824575, Dzl3 (e.g., TRC-093), D93,ATL-1103, XV-615, pSivida, VEGF/rGel, VAR-10200, VAL-566-620-MULTI, TKI,TK-001, STP-601, dry AMD stem cell therapy (e.g., EyeCyte), OpRegen,SMT-D004, SAR-397769, RTU-007, RST-001, RGNX-004, RFE-007-CAI, MC-2002,lycium anti-angiogenic proteoglycan, IXSVEGF, integrin inhibitors,GW-771806, GBS-007, Eos-013, EC-400, dry-AMD therapy (e.g., NeuronSystems), CGEN-25017, CERE-140, AP-202, AC-301, 4-IPP, zinc-monocysteinecomplexes (e.g., Adeona), prinomastat, Neovastat, mecamylamine,CereCRIB, BA-285, ATX-S 10, AG-13958, verteporfin/alphavB3 conjugate,VEGF/rGel, VEGF-saporin, VEGF-R2 antagonist (e.g., Allostera), VEGFinhibitors (e.g., Santen), VEGF antagonists (e.g., Ark), Vangiolux™,Triphenylmethanes (e.g., Alimera), TG-100-801, TG-100-572, TA-106,T2-TrpRS, SU-0879, SHEF-1, rostaporfin (e.g., Photrex™, Purlytin™,SnET2), retino-NPY, PJ-34, PI3K conjugates (e.g., Semafore), PhotoPoint,PAN-90806, Opt-21, OPK-HVB-010, OPK-HVB-004, Ophthalmologicals (e.g.,Cell NetwoRx), OcuXan, NTC-200, NT-502, NOVA-21012, Neurosolve™,neuroprotective (e.g., BDSI), MEDI-548, MCT-355, McEye™, LentiVue™,LYN-002, LX-213, lutetium texaphyrin (e.g., Antrin™), LG-339 inhibitors(e.g., Lexicon), KDR kinase inhibitors (e.g., Merck), ISV-616,INDUS-815C, ICAM-1 aptamer (e.g., Eyetech), GTx-822, GS-102, GranzymeB/VEGF™, gene therapy (e.g., EyeGate), GCS-100 analogue programme,FOV-RD-27, fibroblast growth factor (e.g., Ramot), Panzem SR™, ETX-6991,ETX-6201, EG-3306, D_(Z)-13, disulfiram (e.g., ORA-102), Diclofenac(e.g., Ophthalmopharma), ACU-02, CLT-010, CLT-009, CLT-008, CLT-007,CLT-006, CLT-005, CLT-004, CLT-003 (e.g., Chirovis™), CLT-001, Cethrin™(e.g., BA-210), celecoxib, CD91 antagonist (e.g., Ophthalmophar), CB-42,BNC-4, bestrophin, batimastat, BA-1049, AVT-2, AVT-1, atu012, Apelprogramme (e.g., ApeX-2), anti-VEGF (e.g., Gryphon), AMD ZFPs (e.g.,ToolGen), AM-1101, ALN-VEG01, AK-1003, AGN-211745, ACU-XSP-001 (e.g.,Excellair™), ACU-HTR-028, ACU-HHY-011, ACT-MD (e.g., NewNeural), ABCA4modulators (e.g., Active Pass), A36 (e.g., Angstrom), 267268 (e.g.,SB-267268), 131-TTM-601, vandetanib (e.g., Caprelsa™, Zactima™,Zictifa™), sunitinib malate (e.g., Sutene™, Sutent™), sorafenib (e.g.,Nexavar™), axitinib (e.g., Inlyta™), tivozanib, XL-647, RAF-265,pegdinetanib (e.g., Angiocept™), MGCD-265, icrucumab, foretinib,ENMD-2076, BMS-690514, regorafenib, ramucirumab, plitidepsin (e.g.,Aplidin™), orantinib, nintedanib (e.g., Vargatef™), motesanib,midostaurin, linifanib, telatinib, lenvatinib, elpamotide, dovitinib,cediranib (e.g., Recentin™), JI-101, cabozantinib, brivanib, apatinib,Angiozyme™, X-82, SSR-106462, rebastinib, PF-337210, IMC-3C5, CYC116,AL-3818, VEGFR2 inhibitor (e.g., AB Science), VEGF/rGel (e.g., ClaytonBiotechnologies), TLK-60596, TLK-60404, R84 antibody (e.g., Peregrine),MG-516, FLT4 kinase inhibitors (e.g., Sareum), flt-4 kinase inhibitors,Sareum, DCC-2618, CH-330331, XL-999, XL-820, vatalanib, SU-14813,semaxanib, KRN-633, CEP-7055, CEP-5214, ZK-CDK, ZK-261991, YM-359445,YM-231146, VEGFR2 kinase inhibitors (e.g., Takeda), VEGFR-2 kinaseinhibitors (e.g., Hanmi), VEGFR-2 antagonist (e.g., Affymax), VEGF/rGel(e.g., Targa), VEGF-TK inhibitors (e.g., AstraZeneca), resveratrol,tyrosine kinase inhibitors (e.g., Abbott), tyrosine kinase inhibitors(e.g., Abbott), Tie-2 kinase inhibitors (e.g., GSK), SU-0879, SP-5.2,sorafenib bead (e.g., Nexavar™ bead), SAR-131675, Ro-4383596, R-1530,Pharmaprojects No. 6059, OSI-930, OSI-817, OSI-632, MED-A300,L-000021649, KM-2550, kinase inhibitors (e.g., MethylGene), kinaseinhibitors (e.g., Amgen), Ki-8751, KDR kinase inhibitors (e.g.,Celltech), KDR kinase inhibitors (e.g., Merck), KDR kinase inhibitors(e.g., Amgen), KDR inhibitors (e.g., Abbott), KDR inhibitor (e.g.,LGLS), JNJ-17029259, and IMC-1C11.

In some embodiments, the modulator, e.g. connexin or pannexin channelmodulator, and the ocular treatment agent can be co-formulated forco-administration. In some aspects, the formulation of the modulator,e.g. connexin or pannexin channel modulator, and the ocular treatmentagent can be a pill, a solution, a gel, a pre-filled syringe, a tablet,eye drops, or as part of a particle-based formulation. In some aspects,the compound which acts by a different mechanism to reduce IOP can beRhopressa. In some embodiments, the compound which acts by a differentmechanism to reduce IOP can be Roclatan. In some aspects, the compoundwhich acts by a different mechanism to reduce IOP can be an adenosinemimetic. In some embodiments, the adenosine mimetic can be Trabodenoson(N-Cyclopentyladenosine 5′-nitrate).

In some embodiments, provided are methods of modulating and/orinhibiting the action of a rho kinase and connexin or pannexin channelactivity in a cell or medium. A cell may be in a body in vivo, or in aliving body in vivo, or in vitro. A medium may include an assay medium.The methods may comprise applying to a medium or contacting a cell withan effective amount of a rho kinase inhibitor and a modulator, e.g.connexin or pannexin channel modulator.

In some embodiments, the rho kinase inhibitor may be amino isoquinolylamides or amino benzamidyl amides of deLong et al. (U.S. Pat. No.8,716,310, and herein incorporated by reference), 2H-isoquinolin-1-oneand 3H-isoquinolin-4-one derivatives of Bosanac et al. (U.S. Pat. No.8,809,326), 6-aminoisoquinoline derivatives of deLong (U.S. Patent Publ.2013/0137721), beta and gamma-amino isoquinoline amide compounds andsubstituted benzamide compounds of deLong (U.S. Patent Publ.2014/0249201), the isoquinoline compounds covalently linked to aprostaglandin or a prostaglandin analog of Kopczynski (U.S. Patent Publ.2014/0275161 and U.S. Patent Publ. 2014/0275160), the isoquinolinecompounds of deLong (U.S. Pat. No. 8,034,943), the isoquinolinecompounds of deLong (U.S. Pat. Nos. 7,671,205, 7,470,787, 8,455,513,8,450,344, 8,357,699, 8,455,647, and 8,455,514), Rhopressa, Roclatan, orthe compounds and formulations of Richards (PCT App. No.PCT/US2009/047108).

In some embodiments, provided are methods of treating a disease,disorder, or condition. The methods may comprise administering to asubject a rho kinase and a modulator, e.g. connexin and/or pannexinchannel modulator. The methods may comprise administering to a subject acomposition comprising a rho kinase inhibitor compound and a modulator,e.g. connexin and/or pannexin channel modulator, and a pharmaceuticallyacceptable carrier. Diseases, disorders, or conditions may includeocular diseases or conditions associated with rho kinase activity orocular diseases or conditions affected by rho kinases. For example, thedisease may be selected from the group consisting of glaucoma, Wet AMD,Dry AMD (inflammation), and DME. The combination of a rho kinaseinhibitor with a modulator, e.g. a connexin and/or pannexin channelmodulator, of the present invention may also be useful in decreasingintraocular pressure. Thus, these formulations may be useful in thetreatment of glaucoma, such as hypertensive glaucoma or normatensiveglaucoma. The rho kinase inhibitor and modulator, e.g. connexin and/orpannexin channel modulator can be formulated as distinct species forseparate administrations, or as a mixture for a common administration.For the separate administration of the rho kinase inhibitor, the routeof administration for treating ocular diseases can be topically,interperitonally, intravitrealously, or systemically. For the commonadministration of the rho kinase inhibitor and modulator, the route ofadministration for treating ocular diseases can be topically,interperitonally, intravitrealously, or systemically. In someembodiments, for separate or common administration the formulation ofthe rho kinase inhibitor or its derivatives may be formulated forparenteral delivery by a route such as intravenous, subcutaneous,intramuscular, and intra-articular administration. These formulationsare either liquids or lyophilizates. The liquid or lyophilizedformulations can comprise of 1-50% of a rho kinase inhibitor or itsderivatives, the range of dosages of the modulators as described herein,and remaining ingredients selected from solubilizers, stabilizers,buffers, tonicity modifiers, bulking agents, viscosityenhancers/reducers, surfactants, chelating agents, and adjuvants. Theseingredients are well known to one of ordinary skill in the art.Lyophilized formulations need to be reconstituted prior toadministration. Liquid formulations are optionally diluted withpharmaceutically acceptable diluents such as 5% Dextrose Injection, USPor 0.9% Sodium Chloride Injection, USP. These formulations arepreferably administered by infusion although bolus administration isalso possible.

For the separate administration (coadministration) of the rho kinaseinhibitor and the modulator, e.g. connexin or pannexin channelmodulator, the administrations can be sequential or simultaneous. Forsequential administrations, the rho kinase inhibitor and the connexin orpannexin modulator can be administered within one hour of each other,within one day of each other, within one week of each other, or withinone month of each other. For simultaneous administration(coadministration), the rho kinase inhibitor and the modulator can beadministered together as a mixture, or as a formulation comprising boththe rho kinase inhibitor and the modulator, e.g. connexin or pannexinchannel modulator.

In some aspects, the invention provides methods of modulating rho kinaseactivity and connexin or pannexin channel activity, the methodscomprising contacting a cell with a rho kinase inhibitor, in an amounteffective to modulate rho kinase activity, and contacting a cell with aconnexin and/or pannexin channel modulator, for example, in an amounteffective to modulate connexin and/or pannexin channel activity.

In some aspects, the invention provides methods of reducing intraocularpressure, the methods comprising contacting a cell with a rho kinaseinhibitor and a modulator, e.g. connexin or pannexin channel modulator,as described herein, in an amount effective to reduce intraocularpressure.

For separate or common administration, the dosage range of the rhokinase inhibitor for systemic administration is from about 0.001 toabout 100 mg/kg body weight, preferably from about 0.01 to about 10mg/kg per body weight, most preferably form about 0.05 to about 5 mg/kgbody weight per day. The rho kinase inhibitor dose can be 0.001, 0.002,0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04,0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mg/kg body weight or anyrange between any two of the recited dosages or any dose between any tworecited numbers. For separate or common administration, the dosage rangeof the modulator, e.g. connexin or pannexin channel modulator, is fromabout 1.0 ng/kg body weight to about 10 mg/kg body weight, or from about1 ug/kg body weight to about 5 mg/kg body weight, or from about 50 ug/kgbody weight to about 1 mg/kg body weight. The modulator, e.g. connexinand/or pannexin channel modulator dose can be 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4,8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100 ng/kg body weight, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0,3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100 ug/kg body weight, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mg/kg bodyweight, or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3,6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 g/kg body weight.

For separate or common administration, the formulation may be preparedto provide for rapid or slow release; immediate, delayed, timed, orsustained release; or a combination thereof. Formulations may be in theform of liquids, solutions, suspensions, emulsions, elixirs, syrups,electuaries, drops (including but not limited to eye drops), tablets,granules, powders, lozenges, pastilles, capsules, gels, ointments,creams, lotions, oils, foams, sprays, mists, or aerosols. As anadditional embodiment, the pharmaceutical formulation can be containedwithin, delivered by, or attached to contact lenses that are placed onthe eye.

Articles of Manufacture/Kits of Combinations of Connexin or PannexinModulators with Rho Kinase Inhibitors or Other Agents

In another embodiment of the invention, an article of manufacture, or“kit”, containing materials useful for treating the diseases anddisorders described above is provided. The kit comprises a containercomprising a rho kinase inhibitor and a modulator, e.g. connexin and/orpannexin channel modulator. The kit may further comprise a label orpackage insert, on or associated with the container. The term “packageinsert” is used to refer to instructions customarily included incommercial packages of therapeutic products, that contain informationabout the indications, usage, dosage, administration, contraindicationsand/or warnings concerning the use of such therapeutic products.Suitable containers include, e.g., bottles, vials, syringes, blisterpack, etc. The container may be formed from a variety of materials suchas glass or plastic. The container may hold a rho kinase inhibitor and amodulator, e.g. connexin and/or pannexin channel modulator, or aformulation thereof which is effective for treating the condition andmay have a sterile access port (e.g., the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). At least one active agent in thecomposition is a modulator, e.g. a connexin and/or pannexin channelmodulator. The label or package insert indicates that the composition isused for treating the condition of choice, such as hypertensive ornormatensive glaucoma, DME, ocular fibrosis, wet AMD, or dry AMD, or anyof the ocular disorders described above. The label or package insert mayalso indicate that the composition can be used to treat other disorders.Alternatively, or additionally, the article of manufacture may furthercomprise a second container comprising a pharmaceutically acceptablebuffer, such as bacteriostatic water for injection (BWFI),phosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes.

The kit may further comprise directions for the administration of themodulator, e.g. connexin and/or pannexin channel modulator, and, ifpresent, the rho kinase inhibitor or other agent which acts on aseparate mechanism from connexin and/or pannexin channel modulation toreduce TOP as described herein. For example, if the kit comprises afirst composition comprising a connexin or pannexin modulator, and asecond pharmaceutical formulation, the kit may further comprisedirections for the simultaneous, sequential or separate administrationof the first and second pharmaceutical compositions to a patient in needthereof.

In certain other embodiments wherein the kit may comprise a modulator,e.g. connexin and/or pannexin channel modulator, and a rho kinaseinhibitor or other agent which acts on a separate mechanism fromconnexin and/or pannexin channel modulation, e.g., to reduce TOP asdescribed herein, the kit may comprise a container for containing theseparate compositions such as a divided bottle or a divided foil packet,however, the separate compositions may also be contained within asingle, undivided container. Typically, the kit comprises directions forthe administration of the separate components. The kit form isparticularly advantageous when the separate components are preferablyadministered in different dosage forms (e.g., ocular and parenteral),are administered at different dosage intervals, or when titration of theindividual components of the combination is desired by the prescribingphysician.

The invention includes an article of manufacture comprising packagingmaterial containing one or more dosage forms containing modulator, e.g.connexin and/or pannexin channel modulators and a rho kinase inhibitoror other agent which acts on a separate mechanism from connexin and/orpannexin channel modulation to reduce TOP as described herein, whereinthe packaging material has a label that indicates that the dosage formcan be used for a subject having or suspected of having or predisposedto any of the diseases, disorders and/or conditions described orreferenced herein. Such dosage forms include, for example, tablets,capsules, solutions and suspensions for parenteral and ocular deliveryforms and formulations.

In some aspects, the kit can comprise the rho kinase inhibitorcompartmentalized in a different compartment than the modulator, e.g.connexin and/or pannexin channel modulator, for separate administration.The compartments can be separate vials, separate syringes, separatecontact lenses, separate solutions, or separate particles-infused withthe agents as described above. In some aspects, the rho kinase inhibitorcan be compartmentalized for delivery to the front of the eye while theconnexin and/or pannexin channel modulator, e.g., can becompartmentalized for delivery to the posterior segments of the eye(retina, choroid, RPE, Bruch's membrane). In some aspects, the connexinand/or pannexin channel modulator, e.g., can be compartmentalized in theform of nanoparticle or microparticle form as described herein forsustained delivery to the back of the eye while the rho kinase inhibitorcan be compartmentalized for immediate or sustained delivery to thefront of the eye in the form of eye drops or contact lens infused withthe rho kinase inhibitor.

In one aspect, the kit may further comprise a third container comprisinga pharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

In some aspects, the first and second (and optionally, third)compositions of the kit can be administered in combination, can beadministered simultaneously, can be administered separately, can beadministered sequentially, or can be administered in a sustained manner.

Iontophoresis

The connexin modulator formulations of this invention may also beadministered by iontophoresis, a non-invasive procedure in which drugpenetration is enhanced by applying a small electric current. Themodulator, e.g. connexin and/or pannexin channel modulator is appliedwith an electrode carrying the same charge as the drug, and the groundelectrode, which is of the opposite charge, is placed elsewhere on thebody to complete the circuit. The modulator helps to conduct the currentthrough the tissue. Transcleral iontophoresis can be used to delivertherapeutic levels of bioactive polynucleotides and peptides to theretina and the choroid.

Doses, Amounts and Concentrations

As will be appreciated, the dose of modulator, e.g. gap junction channelmodulator, such as peptide 5, and/or an analogue thereof, compounds offormula I, for example tonabersat, and analogs of any of the foregoingcompounds, and/or a pannexin modulator, e.g., probenecid and an analoguethereof, and/or a synthetic mimetic peptide blocker of pannexin 1, e.g.,¹⁰Panx1, or an analogue thereof administered, the period ofadministration, and the general administration regime may differ betweensubjects depending on such variables as the target site to which it isto be delivered, the severity of any symptoms of a subject to betreated, the type of disorder to be treated, size of unit dosage, themode of administration chosen, and the age, sex and/or general health ofa subject and other factors known to those of ordinary skill in the art.

Data obtained from cell culture assays and animal studies can be used informulating a range of dosages for use in humans. The modulator dosagemay vary within this range depending upon the dosage form employed andthe route of administration utilized. For any agent used in the methodof the invention, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose may be formulated in cellcultures or animal models to achieve a cellular concentration range thatincludes the IC50 (i.e., the concentration of the test compound thatachieves a half-maximal inhibition of symptoms) as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition. (See, e.g., Fingl et al., 1975, In: ThePharmacological Basis of Therapeutics, Ch. 1, p. 1). The dosage can bedetermined from the concentration of the amount administered, expectedmass of the animal model tested (200-300 g per rat for adult Wistarrats), to determine the dose in units of mg/kg from concentration(micromolar) administered or amount (mg) administered.

Examples of effective doses that may be used for the treatment of oculardisorders such as glaucoma ocular hypoxia, retinal perfusion impairment,AMD, DME, eye fibrosis and/or ocular neuropathy, or any other oculardisorder described herein are described and claimed herein. In someaspects, the therapeutically effective amount of the modulator, e.g.connexin modulator, and/or pannexin channel modulator, for example aconnexin 43 or connexin 43 hemichannel modulator, which is effective totreat ocular disorders, for example, ocular hypertension, glaucoma, AMD,DME, ocular fibrosis, glaucomatous ocular neuropathy, RGC loss,impairment of choridal perfusion, choroidal inflammation, choroidaloverperfusion, and/or choriocapillaris dropout is a concentration ofabout 0.001 to about 1.0 microgram/ml, or from about 0.001 to about 0.01mg/ml, or from about 0.1 mg/mL to about 100 mg/mL, or more, or any rangebetween any two of the recited dosages or any dose between any tworecited numbers. The dose can be 0.001, 0.002, 0.003, 0.004, 0.005,0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100 mg/ml or any range between any two of the reciteddosages or any dose between any two recited numbers. In someembodiments, the therapeutically effective amount of the modulator, e.g.connexin and/or pannexin channel modulating agent protein modulatingagent is present at a concentration ranging from about 0.5 to about 50mg/mL. In some embodiments, the modulating agent is present at aconcentration ranging from about 0.3 to about 30 mg/mL. In someembodiments, the modulating agent is present at a concentration rangingfrom about 0.1 or 1.0 to about 10 mg/mL. In some embodiments, themodulating agent is present at a concentration ranging from about 0.1 or1.0 to about 0.3 or 3.0 mg/mL. In some embodiments, the modulating agentis present at a concentration of about 3.0 mg/mL. In any of theseaspects the modulating agent may be a connexin protein antisenseoligonucleotide. When the modulating agent is a modified antisenseoligonucleotide, e.g., a backbone-modified oligonucleotide, orchemically modified oligonucleotide for increased half-life, theabove-noted dose concentrations may be the same, or may be decreased orincreased as appropriate based on potency and specificity, for example.In any of these aspects, the carrier (vehicle) may be a pharmaceuticallyacceptable carrier.

In some aspects the modulator, e.g. connexin modulator and/or pannexinchannel modulator may be administered at a therapeutically effectivedose between about 0.001 to about 100 mg/kg, between about 0.001 toabout 0.01 mg/kg, between about 0.01 to about 0.1 mg/kg, between 0.1 toabout 1 mg/kg, between about 1 to about 10 mg/kg, or between about 10 toabout 100 mg/kg, or any range between any two recited dosages or anydose between any two recited dosages. In some aspects, the dose can be0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mg/ml or anyrange between any two of the recited dosages or any dose between any tworecited numbers.

Although each of the therapeutically effective concentrations, amountsor doses for use with this invention as exemplified above may be usefulfor modified or unmodified nucleic acid modulators, modified orunmodified peptide or peptidomimetic modulators, or small moleculemodulators, in some aspects of this invention, for each of thetherapeutically effective concentrations, amounts or doses describedabove, the dose of a peptide or peptidomimetic connexin modulator orpannexin modulator may be between 1/10 to 1/100, or between 1/100, to1/1000 of any of the recited concentrations, amounts or doses. Inaddition, the therapeutically effective concentrations, amounts or dosefor a modified modulator, e.g. connexin modulator, pannexin modulator,or pannexin channel modulator may, in some aspects, be between 1/10 to1/100, or between 1/100, to 1/1000 of the recited concentrations,amounts or doses, or any range between any two recited dosages or anydose between any two recited dosages.

It should be appreciated that administration may include a single dailydose, administration of a number of discrete divided doses, orcontinuous administration, as may be appropriate. By way of example,unit doses may be administered once or more than once per day, forexample 1, 2, 3, 4, 5 or 6 times a day to achieve a desired total dailydose. By way of example, a unit dose of a gap junction channelmodulator, e.g., such as peptide 5, and/or an analogue thereof,compounds of formula I, for example tonabersat, and analogs of any ofthe foregoing compounds, and/or a pannexin modulator, e.g., probenecidand an analogue thereof, and/or a synthetic mimetic peptide blocker ofpannexin 1, e.g., ¹⁰Panx1, or an analogue thereof may be administered ina single daily dose or a number of discrete doses, or continuously toachieve a daily dose of approximately 0.1 to 10 mg, 10 to 100 mg, 100 to1000 mg, 1000 to 2000 mg, or 2000 mg to 5000 mg, 0.1 to approximately2000 mg, approximately 0.1 to approximately 1000 mg, approximately 1 toapproximately 500 mg, approximately 1 to approximately 200 mg,approximately 1 to approximately 100 mg, approximately 1 toapproximately 50 mg, or approximately 1 to approximately 25 mg, or anyrange between any two recited dosages or any dose between any tworecited dosages.

By way of further example, a unit dose of a modulator, e.g. gap junctionchannel modulator, such as peptide 5, tonabersat and/or an analogue ofeither, and/or a pannexin modulator, e.g., probenecid and an analoguethereof, and/or a synthetic mimetic peptide blocker of pannexin 1, e.g.,¹⁰Panx 1, or an analogue thereof may be administered once or more thanonce a day (for example 1, 2, 3, 4, 5 or 6, typically 1 to 4 times aday), such that the total daily dose is in the range (for a 70 kg adult)of approximately 1 to approximately 1000 mg, for example approximately 1to approximately 500 mg, or 500 mg to 1000 mg, 1000 to 2000 mg, or 2000mg to 5000 mg, or any range between any two recited dosages or any dosebetween any two recited dosages. For example, a gap junction channelmodulator, such as peptide 5, and/or an analogue thereof, compounds offormula I, for example tonabersat, and analogs of any of the foregoingcompounds, and/or a pannexin modulator, e.g., probenecid and an analoguethereof, and/or a synthetic mimetic peptide blocker of pannexin 1, e.g.,¹⁰Panx1, or an analogue thereof may be administered to a subject at adose range of approximately 0.01 to approximately 15 mg/kg/day, forexample approximately 0.1 to approximately 6 mg/kg/day, for exampleapproximately 1 to approximately 6 mg/kg/day, for example, 6 mg/kg/dayto 100 mg/kg/day or any range between any two recited dosages or anydose between any two recited dosages. In one embodiment, tonabersat maybe administered orally once a day at a dose of approximately 2 mg toapproximately 40 mg.

In one embodiment, the dose of compounds of formula I, for exampletonabersat, and analogs of any of the foregoing compounds isapproximately 0.001 micromolar to 0.1 micromolar, 0.1 micromolar and upto approximately 200 micromolar at the site of action, or higher, withinthe circulation to achieve those concentrations at the site of action.By way of example, the dose may be (but not limited to) a finalcirculating concentration of about 0.001, 0.002, 0.003, 0.004, 0.005,0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340,350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,490, or 500 micromolar, or any range between any two recitedconcentrations, or any concentration between any two recited numbers.Further examples of doses expected to block hemichannels but not touncouple gap junctions are described in O'Carroll et al, 2008, hereinincorporated by reference. In some embodiments, tonabersat may be usedat a lower dose, for example, 0.001 to 20 micromolar. A low dose can be0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 micromolar. It was surprisingly determined,for example, that lower doses of tonabersat are useful to inhibit Cx43hemichannel mediated ATP release during reperfusion. The use of suchlower doses of tonabersat in the methods and uses of this invention,alone or in combination with a pannexin modulator, are also advantageousin reducing side effects of tonabersat.

In other aspects, the time-dependent action of tonabersat with respectto transiently counteracting a rise in Cx43 following injury, and hencede novo formation of GJ hemichannels could be utilised for controlledtransient modulation of Connexin 43 channels, e.g., following injurywhile minimizing the complete removal of the ‘spatial-buffering’ effectto prevent the accumulation of toxic metabolites and to synchronisetissues following injury, particularly in the central nervous system.

In one embodiment, the dose of a modulator, e.g. a gap junction channelmodulator, such as peptide 5 and/or an analogue thereof, and/or apannexin channel modulator, e.g., probenecid and an analogue thereof,and/or a synthetic mimetic peptide blocker of pannexin 1, e.g., ¹⁰Panx1,or an analogue thereof is approximately 0.001 micromolar and up toapproximately 200 micromolar, or 200 to 2000 or 5000 micromolar at thesite of action, or higher within the circulation to achieve thoseconcentrations at the site of action. By way of example, the dose may be(but not limited to) a final circulating concentration of about 1, 5,10, 20, 50, 100, 200, 250, 500, 1000, 2000, 3000, 4000, or 5000micromolar, or any range between any two recited dosages or any dosebetween any two recited dosages. Doses of peptide 5 effective to blockhemichannels but not to uncouple gap junctions are discussed inO'Carroll et al, 2008. In some embodiments, tonabersat may be used at alower dose, for example, 1 to 20 micromolar, 1 to 50 micromolar, 20 to30, 30 to 40 or 40 to 50 micromolar. A low dose can be 0.001, 0.002,0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04,0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 micromolar. It was surprisingly determined, for example, thata lower dose of tonabersat inhibits Cx43 hemichannel mediated ATPrelease during ischemia-reperfusion. It was surprisingly determined thatcertain doses of tonabersat inhibit hemichannels formatting, therebytreating reperfusion following ischemia.

In some embodiments, a suitable therapeutically effective dose of amodulator, e.g. a connexin modulator or pannexin modulator or channelmodulators thereof, may be at least about 1.0 mg/mL of the modulator,e.g. connexin modulator or pannexin or channel modulator. In someembodiments, the therapeutically effective dose of the modulator, e.g.connexin modulator and/or pannexin modulator, and channels thereof, maybe from about 0.001 mg/mL to 0.01 mg/mL, from about 0.01 mg/mL to about0.1 mg/mL, or from about 0.1 mg/mL to about 100 mg/mL. In someembodiments, the suitable therapeutically effective dose of connexinmodulator or pannexin modulator may be about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0,11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0,23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0,35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0,47.0, 48.0, 49.0, 50.0, 52.5, 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0,72.5, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 90.0, 92.5, 95.0, 97.5, orabout 100.0 ug/mL, or any range or subrange between any two of therecited doses, or any dose falling within the range of about 0.1 toabout 100 ug/mL. In some embodiments, the suitable therapeuticallyeffective dose of an anti-connexin agent may be about 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0,9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0,21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0,33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0,45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 52.5, 55.0, 57.5, 60.0, 62.5, 65.0,67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 90.0, 92.5, 95.0,97.5, or about 100.0 mg/mL, or any range or subrange between any two ofthe recited doses, or any dose falling within the range of about 0.1 toabout 100 mg/mL. In some embodiments, the modulator, e.g. connexinmodulator and/or pannexin channel modulator, is present at aconcentration ranging from about 0.5 to about 50 mg/mL. In otherembodiments, the modulator, e.g. connexin modulator and/or pannexinchannel modulator, is present at a concentration ranging from about 0.3to about 30 mg/mL. In other embodiments, the modulator is present at aconcentration ranging from about 0.1 or 1.0 to about 10 mg/mL. In otherembodiments, the modulator is present at a concentration ranging fromabout 0.1 or 1.0 to about 0.3 or 3.0 mg/mL. In other embodiments, amodulator, e.g., a connexin modulator or pannexin modulator, such as aconnexin 43 modulating agent, and/or a connexin 45 modulating agent ispresent at a concentration of about 3.0 mg/mL. In any of these aspectsthe modulator, e.g. connexin modulating agent may be a connexin 43, orconnexin 45 modulating agent, for example, a connexin 43 or connexin 45antisense oligonucleotide, preferably a connexin 43 modulating agent,for example, a connexin 43 antisense oligonucleotide. When the modulatoris a modified connexin or pannexin antisense oligonucleotide theabove-noted dose concentrations may be increased by from about 2- toabout 10-fold, for example. When the modulator is a modified orunmodified peptide or peptidomimetic, the dose may be decreased by 10,100, or 1000 fold.

In certain embodiments, the connexin or pannexin modulators, forexample, the connexin 43 modulator may be administered at about 0.001micromolar (μM) or 0.05 μM to about 200 μM, or up to 300 μM or up to1000 μM or up to 2000 μM or up to 3200 μM or more, for example up toabout 10 mM, 20 mM, or 30 mM final concentration at the treatment siteand/or adjacent to the treatment site, and any doses and dose rangeswithin these dose numbers. In one embodiment, the connexin modulator(anti-connexin agent) composition is applied at greater than about 1000μM. Preferably, the antisense polynucleotide composition is applied atabout 1000 μM to about 10 mM final concentration, more preferably, theanti-connexin agent composition is applied at about 3 mM to about 10 mMfinal concentration, and more preferably, the anti-connexin agentcomposition is applied at about 1-3 mM to about 5-10 mM finalconcentration. The connexin modulator concentration can be 0.001, 0.002,0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04,0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 micromolar; or 0.001, 0.002,0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04,0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 millimolar, or any rangebetween any two of the recited dosages or any dose between any tworecited numbers.

Additionally, modulator, e.g. connexin and pannexin channel modulators,for example, connexin 43 modulators may be present in the formulation atabout 1 μM to about 20 μM final concentration, and alternatively theconnexin 43 modulator is present at about 5 μM to about 20 μM finalconcentration, or at about 10 to about 15 μM final concentration. Incertain other embodiments, the modulator is present at about 10 μM finalconcentration. In yet another embodiment, the modulator is present atabout 1-15 μM final concentration. In other embodiments, the modulatoris present at about 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90μM, 100 μM, 10-200 μM, 200-300 μM, 300-400 μM, 400-500 μM, 500-600 μM,600-700 μM, 700-800 μM, 800-900 μM, 900-1000 or 1000-1500 μM, or 1500μM-2000 μM, 2000 μM-3000 μM, 3000 μM-4000 μM, 4000 μM-5000 μM, 5000μM-6000 μM, 6000 μM-7000 μM, 7000 μM-8000 μM, 8000 μM-9000 μM, 9000μM-10,000 μM, 10,000 μM-11,000 μM, 11,000 μM-12,000 μM, 12,000 μM-13,000μM, 13,000 μM-14,000 μM, 14,000 μM-15,000 μM, 15,000 μM-20,000 μM,20,000 μM-30,000 μM, 30,000 μM-50,000 μM, or greater, or any range orsubrange between any two of the recited doses, or any dose fallingwithin the range of from about 20 μM to about 50,000 μM.

Still other dosage levels between about 1 nanogram (ng)/kg and about 1mg/kg body weight per day of each of the modulators described herein. Incertain embodiments, the dosage of each of the subject compounds willgenerally be in the range of about 1 ng to about 1 microgram per kg bodyweight, about 1 ng to about 0.1 microgram per kg body weight, about 1 ngto about 10 ng per kg body weight, about 10 ng to about 0.1 microgramper kg body weight, about 0.1 microgram to about 1 microgram per kg bodyweight, about 20 ng to about 100 ng per kg body weight, about 0.001 mgto about 0.01 mg per kg body weight, about 0.01 mg to about 0.1 mg perkg body weight, or about 0.1 mg to about 1 mg per kg body weight. Incertain embodiments, the dosage of each of the subject compounds willgenerally be in the range of about 0.001 mg to about 0.01 mg per kg bodyweight, about 0.01 mg to about 0.1 mg per kg body weight, about 0.1 mgto about 1 mg per kg body weight. If more than one modulator, e.g.connexin and/or pannexin channel modulator, is used, the dosage of eachmodulator need not be in the same range as the other. For example, thedosage of one connexin or pannexin channel modulator may be betweenabout 0.01 mg to about 10 mg per kg body weight, and the dosage ofanother connexin or pannexin channel modulator may be between about 0.1mg to about 1 mg per kg body weight, 0.1 to about 10, 0.1 to about 20,0.1 to about 30, 0.1 to about 40, or between about 0.1 to about 50 mgper kg body weight. The dosage may also be about 0.001, 0.002, 0.003,0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05,0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100 mg/kg body weight or any range orsubrange between any two of the recited doses, or any dose fallingwithin the range of from about 0.001 to about 100 mg per kg body weight.

In one embodiment, the combined use of one or more modulators, e.g.connexin, hemichannel, pannexin or pannexin channel modulatorpolynucleotides or one or more connexin or pannexin modulator peptidesor peptidomimetics reduces the effective dose of any such agent comparedto the effective dose when said agent administered alone. In certainembodiments, the effective dose of the agent when used in combination isabout 1/15 to about ½, about 1/10 to about ⅓, about ⅛ to about ⅙, about⅕, about ¼, about ⅓ or about ½ the dose of the agent when used alone. Inanother preferred embodiment, the combined use of one or moreanti-connexin polynucleotides and one or more anti-connexin peptides orpeptidomimetics, or other anti-connexin agents in combination witheither or both, reduces the frequency in which said agent isadministered compared to the frequency when said agent is administeredalone. Thus, these combinations allow the use of lower and/or fewerdoses of each agent than previously required to achieve desiredtherapeutic goals.

In one embodiment, the dose of a connexin or pannexin modulator peptideor peptidomimetic may be 10, 100 or 1000 fold lower than any of therecited doses set forth herein.

Conveniently, the modulator is administered in a sufficient amount todownregulate expression of its respective protein. The connexinmodulator, for example, a connexin 43 or 45 modulator is administered ina sufficient amount to downregulate expression of connexin 43 ormodulate gap junction formation or connexon opening for at least about0.5 to 1 hour, at least about 1-2 hours, at least about 2-4 hours, atleast about 4-6 hours, at least about 6-8 hours, at least about 8-10hours, at least about 12 hours, or at least about 24 hourspost-administration.

The doses of the connexin or pannexin channel modulator, such as theconnexin 43 or 45 modulator, may be administered in single or dividedapplications. The doses may be administered once, or application may berepeated. Typically, application will be repeated weekly, biweekly, orevery 3 weeks, every month, or every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or every 24 months or moreas needed to prevent, slow, or treat ocular neuropathy, or any ocularcondition described herein. The dose may be repeated, and/or increasedor decreased in the event that neuronal loss increases or decreases.Doses may also be applied every 12 hours to 7 days apart, or more. Forexample, doses may be applied 12 hours, or 1, 2, 3, 4, 5, 6, or 7 daysapart, or at any time interval falling between any two of these times,or between 12 hours and 7 days. The connexin 43 modulator may beadministered for up to four, six, eight, ten, twelve, fourteen, sixteen,eighteen, twenty, twenty-two, twenty-four or twenty-six weeks. For someindications, such as certain ocular uses, more frequent dosing, up tohourly may employed.

Kits, Medicaments and Articles of Manufacture

Optionally, one or more modulators, e.g. connexin or pannexin modulatorpolynucleotides and/or one or more the connexin or pannexin modulatorpeptides or peptidomimetics and/or other anti-connexin agents such as agap junction or hemichannel phosphorylation agent or connexincarboxy-terminal polypeptide, or small molecule modulators, alone or incombinations of any of the modulating agents, or other resistant woundhealing agents, may also be used in the manufacture of the medicament,or in a kit. Suitable anti-connexin protein modulating agents,polynucleotides or peptides may be anti-connexin 43, 30 or 26 modulatingagents, polynucleotides or peptides. In some aspects any of theseconnexin modulators is a modulator of Cx31.1, Cx36, Cx37, Cx40, Cx45,Cx50, Cx57 or any other connexin in the eye or blood vessels.

In one aspect, the invention provides an article of manufacture or kitcomprising one or more compositions or formulations described. Forexample, the kit may include a pharmaceutical formulation comprising aneffective amount of one or more the connexin or pannexin modulatorpolynucleotides and/or one or more connexin or pannexin modulatorpeptides or peptidomimetics and/or other anti-connexin agents, such as agap junction or hemichannel phosphorylation agent or connexincarboxy-terminal polypeptide, alone or in combinations of any of theconnexin or pannexin modulator, or other ocular treatment agents. Insome embodiments, the kit may include a pharmaceutical formulationcomprising an effective amount of one or more anti-connexin 43polynucleotides and/or one or more anti-connexin 43 peptides orpeptidomimetics and/or other anti-connexin agents, such as a gapjunction or hemichannel phosphorylation agent or connexincarboxy-terminal polypeptide, alone or in combinations of any of theanti-connexin 43 modulating agents, or other ocular treatment agents. Insome aspects any the kit may comprise is a modulator of Cx26, Cx30,Cx31.1, Cx36, Cx37, Cx40, Cx45, Cx50, Cx57 or any other connexin in theeye or blood vessels.

A kit may comprise one or more pharmaceutical compositions, in separatevessels, or a partitioned vessel, together with packaging andinstructions for use. The kit may also comprise a pharmaceuticallyacceptable carrier. In some embodiments the kit may also includecomponents for administering the pharmaceutical compositions, forexample, a syringe, needle, microneedle, a loadable implant, or aniontophoresis device. The connexin or pannexin modulator and oculartreatment partners as described herein can be dosed independently or byuse of different fixed combinations with distinguished amounts of thecombination partners (a) and (b), i.e. simultaneously, separately orsequentially, whether in pharmaceutical form or dressing/matrix form orboth. A parts of the kit can then, for example, be administeredsimultaneously or chronologically staggered, that is at different timepoints and with equal or different time intervals for any part of thekit of parts.

Articles of manufacturer are also provided, comprising a vesselcontaining a compound, composition or formulation of the invention asdescribed herein and instructions for use for the treatment of asubject. For example, in another aspect, the invention includes anarticle of manufacture comprising a vessel containing a therapeuticallyeffective amount of one or more connexin or pannexin modulatorpolynucleotides and/or one or more connexin or pannexin modulatorpeptides or peptidomimetics and/or other anti-connexin agents, such as agap junction or hemichannel phosphorylation agent or connexincarboxy-terminal polypeptide, alone or in combinations of any of theanti-connexin protein modulating agents, or other resistant woundhealing agents, together with instructions for use, including use forthe treatment of a subject. Suitable anti-connexin protein modulatingagents, polynucleotides or peptides may be connexin 43 or 45 modulatingagents, polynucleotides or peptides.

In some aspects the article of manufacture may comprise a matrix thatcomprises one or more connexin or pannexin modulator peptides orpeptidomimetics or other ocular treatment agents or anti-connexinagents, such as a gap junction or hemichannel phosphorylation agent orconnexin carboxy-terminal polypeptide, alone or in combinations of anyof the anti-connexin 43 modulating agents, or other resistant woundhealing agents, Suitable connexin or pannexin modulator agents,polynucleotides or peptides may be anti-connexin 43 or 45 modulatingagents, polynucleotides or peptides.

Connexin Modulators

Connexin modulators described herein, e.g., connexin 43 or connexin 45modulators, modulate cellular communication (e.g. cell to cell), by, forexample, blocking or inhibiting the transport of molecules into and outof cells. Connexin modulators affect transmission of molecules betweenthe cell cytoplasm and the periplasmic or extracellular space. Suchmodulators are generally targeted to hemichannels (also calledconnexons), which may be independently involved in the exchange of smallmolecules between the cell cytoplasm and an extracellular space ortissue. Thus, a compound provided herein may directly or indirectlyreduce coupling between cells (via gap junctions) or between a cell andan extracellular space or tissue (via hemichannels), and the modulationof transport of molecules from a cell into an extracellular space iswithin the scope of certain compounds and embodiments of the invention.In some aspects the connexin modulator is a modulator of Cx26, Cx30,Cx31.1, Cx36, Cx37, Cx40, Cx 43, Cx45, Cx50, Cx57 or any other connexinin the eye or blood vessels.

Any modulator that is capable of eliciting a desired inhibition of thepassage (e.g., transport) of molecules through a gap junction orconnexin hemichannel in ocular blood vessels may be used in embodimentsof the invention. Any connexin agents that modulates the passage ofmolecules through a gap junction or connexin hemichannel are alsoprovided in particular embodiments (e.g., those that modulate, block orlessen the passage of molecules from the cytoplasm of a cell into anextracellular space or adjoining cell cytoplasm). Such anti-connexinagents may modulate the passage of molecules through a gap junction orconnexin hemichannel with or without gap junction uncoupling (blockingthe transport of molecules through gap junctions). Such compoundsinclude, for example, binding proteins, polypeptides, and other organiccompounds that can, for example, block the function or activity of a gapjunction or a hemichannel in whole or in part. In some embodiments, theanti-connexin modulator may be an anti-connexin 43 modulator or ananti-connexin 45 modulator. In some aspects the connexin modulator is amodulator of Cx26, Cx31.1, Cx36, Cx37, Cx40, Cx50, Cx57 or any otherconnexin in the eye or blood vessels.

Certain connexin modulators, such as connexin 43 or connexin 45modulators, provide downregulation of connexin expression (for example,by downregulation of mRNA transcription or translation) or otherwisedecrease or inhibit the activity of the connexin protein, connexinhemichannels or gap junctions. In the case of downregulation, this willhave the effect of reducing direct cell-cell communication by gapjunctions, or exposure of cell cytoplasm to the extracellular space byhemichannels, at the site at which connexin expression is downregulated.

In certain embodiments, an anti-connexin agent prevents, decreases oralters the activity or function of a hemichannel or a gap junction. Asused herein, modulation of the gap junction activity or function by theanti-connexin agent may include the closing of gap junctions, closing ofhemichannels, and/or passage of molecules or ions through gap junctionsand/or hemichannels.

Pannexin or connexin modulators include anti-connexin, or anti-pannexinpolynucleotides, such as antisense polynucleotides or oligonucleotides,for example, connexin 43 oligodeoxynucleotides and other polynucleotides(such as polynucleotides having siRNA or ribozyme functionalities), aswell as antibodies and binding fragments thereof that bind connexinprotein and a pannexin, and pannexin or connexin peptides andpolypeptides, including peptidomimetics and peptide analogs of connexinthat modulate hemichannel or gap junction activity or function, andother gap junction blocking agents and gap junction proteinphosphorylating agents. Connexin protein peptides and polypeptides may,for example, bind to connexin protein to inhibit its function, or mayinhibit connexin function by mimicking regions of connexin protein toinhibit or disrupt its binding to other gap junction proteins.

Pannexin peptides and polypeptides may, for example, inhibit or disruptpannexin channels.

In other embodiments, the connexin modulators are anti-connexin 43peptides or peptidomimetics, e.g., anti-connexin 43 hemichannel blockingpeptides or peptidomimetics, for example, modified or unmodifiedpeptides or peptidomimentics comprising connexin extracellular domains,transmembrane regions, and connexin carboxy-terminal peptides). Theanti-connexin hemichannel blocking peptides or peptidomimetics may bemodified or unmodified. The anti-connexin hemichannel blocking peptidesor peptidomimetics are made chemically, synthetically, or otherwisemanufactured.

In some embodiments the connexin modulators of this invention includeanti-connexin peptides or peptidomimetics, e.g., connexin 43 peptides orpeptidomimetics, for example, any of the peptides described herein,including peptides comprising a portion of an extracellular domain of aconnexin, and peptides comprising a portion of a carboxy-terminalportion of a connexin useful in the methods of this invention, which istherapeutically effective, for example, effective for healing any of theneuropathic ocular disorders described herein. In some aspects, thetherapeutically effective modified or unmodified peptide orpeptidomimetic comprises a portion of an extracellular or transmembranedomain of a connexin, such as connexin 43.

Methods of synthesizing antibodies and binding fragments as well aspeptides and polypeptides, including peptidomimetics and peptide analogscan also be performed using suitable methods. See e.g. Lihu Yang et al.,Proc. Natl. Acad. Sci. U.S.A., 1; 95(18): 10836-10841 (Sep. 1 1998);Harlow and Lane (1988) “Antibodies: A Laboratory Manuel” Cold SpringHarbor Publications, New York; Harlow and Lane (1999) “Using Antibodies”A Laboratory Manuel, Cold Spring Harbor Publications, New York.

Connexin Polynucleotides and Oligonucleotides

In some aspects of this invention, the connexin modulator is a peptideor peptidomimetic. The connexin modulator may comprise, for example, aconnexin 43 or connexin 45 peptide or peptidomimetic, preferably aconnexin 43 peptide or peptidomimetic. In some aspects the connexinpeptide or peptidomimetic may be a Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40,Cx50, Cx57 peptide or peptidomimetic. In some aspects, the connexinmodulator can include or exclude any of the foregoing.

In some embodiments, the connexin 43 modulator may comprise, forexample, SEQ ID NO: 173 (SRPTEKT), SEQ ID NO: 168 (VDCFLSRPTEKT), or SEQID NO:168 conjugated to two dodecyl groups at the N-terminus, through alinker. The peptide may contain one or more modified amino acids, aminoacid analogs, or may be otherwise modified, for example, conjugated orbound to cell internalization transporter.

In another non-limiting but preferred embodiment, an anti-connexincompound comprises a peptide comprising an amino acid sequencecorresponding to a portion of a transmembrane region of a connexin, suchas connexin 43 or connexin 45, or Cx26, Cx31.1, Cx36, Cx37, Cx40, Cx50,Cx57 or any other connexin in the eye or blood vessels. In particularnon-limiting embodiments, the anti-connexin compound is a peptide havingan amino acid sequence that comprises a peptide having an amino acidsequence that comprises about 3 to about 30 contiguous amino acids ofthe connexin, e.g., connexin 43 or 45 protein sequence, about 5 to about20 contiguous amino acids of the connexin, e.g., connexin 43 or 45protein sequence, a peptide having an amino acid sequence that comprisesabout 8 to about 15 contiguous amino acids of the connexin, e.g.,connexin 43 or 45 protein sequence, or a peptide having an amino acidsequence that comprises about 11, 12, or 13 contiguous amino acids ofconnexin 43 or 45 protein sequence. Other non-limiting embodimentsinclude an anti-connexin compound that is a peptide having an amino acidsequence that comprises at least about 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 20, 25, or 30 contiguous amino acids of the connexin 43 or 45protein sequence. In some aspects, the connexin modulator (anti-connexincompound) can include or exclude any of the foregoing.

In other anti-connexin compounds, mimetic peptides are based on theextracellular domains of connexin 43 corresponding to the amino acids atpositions 37-76 and 178-208 of connexin 43 protein sequence. Thus,certain peptides described herein have an amino acid sequencecorresponding to the regions at positions 37-76 and 178-208 of theconnexin 43 protein sequence. The peptides need not have an amino acidsequence identical to those portions of the connexin 43 proteinsequence, and conservative amino acid changes may be made such that thepeptides retain binding activity or functional activity in the assaysdescribed herein and otherwise known in the art. In other embodiments,mimetic peptides are based on peptide target regions within the connexinprotein other than the extracellular domains (e.g. the portions of theconnexin 43 protein sequence not corresponding to positions 37-76 and178-208).

In some embodiments, the connexin 45 modulator may comprise, forexample, peptides. The peptide may contain one or more modified aminoacids, amino acid analogs, or may be otherwise modified, for example,conjugated or bound to a cell internalization transporter orbioavailability agent.

In a non-limiting but preferred embodiment, a connexin modulatorcomprises a peptide comprising an amino acid sequence corresponding to aportion of a transmembrane region of connexin 45 or a C-terminal regionof connexin 45. In particular non-limiting embodiments, for example, theanti-connexin compound is a peptide having an amino acid sequence thatcomprises about 3 to about 30 contiguous amino acids of the knownconnexin 45 sequence, a peptide having an amino acid sequence thatcomprises about 5 to about 20 contiguous amino acids of the knownconnexin 45 sequence, a peptide having an amino acid sequence thatcomprises about 8 to about 15 contiguous amino acids of the knownconnexin 45 sequence, or a peptide having an amino acid sequence thatcomprises about 11, 12, or 13 contiguous amino acids of the knownconnexin 45 sequence. Other non-limiting embodiments include ananti-connexin compound that is a peptide having an amino acid sequencethat comprises at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 20, 25, or 30 contiguous amino acids of the known connexin 45sequence. In certain anti-connexin compounds provided herein, mimeticpeptides are based on the extracellular domains of connexin 45corresponding to the amino acids at positions 46-75 and 199-228 of theknown connexin 45 sequence. Thus, certain peptide described herein havean amino acid sequence corresponding to the regions at positions 46-75and 199-228 of the known connexin 45 sequence. The peptides need nothave an amino acid sequence identical to those portions of the knownconnexin 45 sequence. and conservative amino acid changes may be madesuch that the peptides retain binding activity or functional activity inthe assays described herein and otherwise known in the art. In otherembodiments, mimetic peptides are based on peptide target regions withinthe connexin protein other than the extracellular domains (e.g. portionsof the known connexin 45 sequence not corresponding to positions 46-75and 199-228). WO2006/134494, disclosing various connexin sequences isincorporated by reference. In some aspects, the connexin modulator caninclude or exclude any of the foregoing.

In some aspects the anti-connexin modulator used in any of the methodsof treatment of this invention is a connexin modulator, for example, aconnexin 45 or connexin 43 modulator. In some embodiments the connexinmodulator is a connexin 43 oligonucleotide or polynucleotide, which maybe, for example, a connexin 43 antisense oligonucleotide orpolynucleotide. The oligonucleotides and polynucleotides of thisinvention are made chemically, synthetically, or otherwise manufactured.In one embodiment the connexin modulator is a connexin protein antisenseoligodeoxynucleotide, whether chemically modified or unmodified. Thisinvention features connexin 43 modulators selected, for example, frommodified or unmodified connexin 43 polynucleotides or oligonucleotides.In some aspects this invention relates to a connexin anti-sense compoundsuch as modified or unmodified connexin 43 antisense oligonucleotidesand modified or unmodified connexin 43 antisense polynucleotides, oroligonucleotides or polynucleotides comprising mixtures of modified andunmodified nucleotides. In some aspects, the connexin 43 antisensecompound used in the methods herein is an antisense oligonucleotidecomprising naturally occurring nucleobases and an unmodifiedinternucleoside linkage. Typically the polynucleotides are singlestranded, but may be double stranded.

Also featured herein are modified or unmodified connexin 43 antisensecompounds comprising a nucleobase sequence selected from SEQ ID NO:1-16.The polynucleotides of this invention include synthesizedpolynucleotides having a length of less than 80 nucleotides, e.g., from12-18 to about 50-80 nucleotides, preferably about 30 nucleotides orless, e.g., from 12 to about 30 nucleotides, and more preferably fromabout 15 to about 30 nucleotides. In one example, the polynucleotide has30 nucleotides. The methods of this invention features, in some aspects,the use of connexin 43 antisense compounds up to 40 nucleotides inlength, for example, 15 to 40 nucleotides in length, comprising anucleotide sequence selected from SEQ ID NO:1-16, or comprising fromabout 8 to 40 nucleotides of SEQ ID NO:17.

In some aspects of this invention, the connexin 43 antisenseoligonucleotide or polynucleotide has at least about 80%, 85%, 90%, 95%,97%, 98%, or 99% homology to a polynucleotide having a sequence selectedfrom SEQ ID NOs: 1 to 16 or a portion of SEQ ID NO:17.

The connexin modulators, including those provided and used in certainembodiments, may comprise one or polynucleotides, selected, for example,from the group consisting of antisense oligonucleotides, antisensepolynucleotides, deoxyribozymes, morpholino oligonucleotides, RNAimolecules, siRNA molecules, PNA molecules, DNAzymes, and 5′-end-mutatedU1 small nuclear RNAs, and analogs of the preceding. These and othercompounds may be used alone or in combination with one more mimetic orother binding peptides.

Featured in this invention are connexin antisense oligonucleotides orpolynucleotides comprising at least one unmodified nucleotide. In oneaspect, the connexin antisense oligonucleotides or polynucleotides maycomprise at least one modified nucleotide, and/or have at least onemodified internucleoside linkage, and/or at least one modified sugarmoiety. The modified internucleoside linkage may be, for example, aphosphorothioate linkage. In some aspects, for example, the connexinpolynucleotide may comprise at least one nucleotide comprising aconformationally strained nucleotide, for example, a locked nucleic acid(LNA) or a bridged nucleic acid (BNA). The locked nucleotide may beselected, from one of the following types, for example: 2′-O—CH₂-4′(oxy-LNA), 2′-CH₂—CH₂-4′ (methylene-LNA), 2′-NH—CH₂-4′ (amino-LNA),2′-N(CH₃)—CH₂-4′ (methylamino-LNA), 2′-S—CH₂-4′ (thio-LNA), and2′-Se—CH₂-4′ (seleno-LNA). In some aspects the modified nucleotide maybe a locked nucleic acid or an unlocked nucleic acid. In some aspectsthe connexin antisense oligonucleotides or polynucleotides are connexin43 antisense oligonucleotides or polynucleotides.

Synthesis of antisense polynucleotides and other anti-connexinpolynucleotides such as RNAi, siRNA, and ribozyme polynucleotides aswell as polynucleotides having modified and mixed backbones can beperformed. See e.g. Stein C. A. and Krieg A. M. (eds), Applied AntisenseOligonucleotide Technology, 1998 (Wiley-Liss).

The antisense polynucleotide may inhibit transcription and/ortranslation of a connexin protein, such as connexin 43 or connexin 45.The antisense polynucleotide is generally antisense to connexin proteinmRNA, for example, connexin 43. Such a polynucleotide may be capable ofhybridizing to connexin protein mRNA and may thus inhibit the expressionof connexin by interfering with one or more aspects of connexin proteinmRNA metabolism including transcription, mRNA processing, mRNA transportfrom the nucleus, translation or mRNA degradation. The antisensepolynucleotide typically hybridizes to the connexin mRNA to form aduplex which can cause direct inhibition of translation and/ordestabilization of the mRNA. Such a duplex may be susceptible todegradation by nucleases. Preferably the polynucleotide is a specificinhibitor of transcription and/or translation from the connexin 43 geneor mRNA, and does not inhibit transcription and/or translation fromother genes or mRNAs. Screening of the polynucleotide sequence in ahuman genome sequence database for specificity may also be performed.The product may bind to the connexin 43 gene or mRNA either (i) 5′ tothe coding sequence, and/or (ii) to the coding sequence, and/or (iii) 3′to the coding sequence.

The antisense polynucleotide may hybridize to part of the connexinprotein mRNA, such as connexin 43 mRNA. Typically the antisensepolynucleotide hybridizes to the ribosome binding region or the codingregion of the connexin protein mRNA. The polynucleotide may becomplementary to a region of the connexin mRNA. For example, thepolynucleotide may be the exact complement of a part of connexin mRNA.However, absolute complementarity is not required and polynucleotideswhich have sufficient complementarity to form a duplex having a meltingtemperature of greater than about 20° C., 30° C. or 40° C. underphysiological conditions are particularly suitable for use in thepresent invention.

Thus the polynucleotide is typically a homologue of a sequencecomplementary to the mRNA. The polynucleotide may be a polynucleotidewhich hybridizes to the connexin protein mRNA under conditions of mediumto high stringency such as 0.03M sodium chloride and 0.03M sodiumcitrate at from about 50° C. to about 60° C.

For certain embodiments of this invention, the polynucleotides of thisinvention include synthesized polynucleotides having a length of lessthan 80 nucleotides, e.g., from 12-18, or 15-18 to about 50-80nucleotides, preferably about 30 nucleotides or less, e.g., from 12 toabout 30 nucleotides, and more preferably from about 15 to about 20, orfrom about 15 to about 30 nucleotides. In one example, thepolynucleotide has 30 nucleotides. The methods of this inventionfeatures, in some aspects, the use of connexin 43 antisense compounds upto 40 nucleotides in length, for example, 15 to 40 nucleotides inlength, comprising a nucleotide sequence selected from SEQ ID NO:1-16,or comprising from about 8 to 40 nucleotides of SEQ ID NO:17.

Alternatively, the antisense polynucleotides may be part of compositionswhich may comprise polynucleotides to more than one connexin protein.Preferably, the connexin protein to which polynucleotides are directedis connexin 43. Suitable exemplary polynucleotides (and ODNs) directedto various connexins are set forth in Table 1.

The polynucleotides for use in the invention may suitably be unmodifiedphosphodiester oligomers, or may be modified. Such oligodeoxynucleotidesmay vary in length. A 30 mer polynucleotide has been found to besuitable, although polynucleotides having 15-30 nucleotides or less arealso suitable.

Many aspects of the invention are described with reference tooligodeoxynucleotides. However it is understood that other suitablepolynucleotides (such as RNA polynucleotides) may be used in theseaspects.

The antisense polynucleotides may be chemically modified. This mayenhance their resistance to nucleases and may enhance their ability toenter cells. For example, phosphorothioate oligonucleotides may be used.Other deoxynucleotide analogs include methylphosphonates,phosphoramidates, phosphorodithioates, N3′P5′-phosphoramidates andoligoribonucleotide phosphorothioates and their 2′-O-alkyl analogs and2′-O-methylribonucleotide methylphosphonates. Alternatively mixedbackbone oligonucleotides (“MBOs”) may be used. MBOs contain segments ofphosphothioate oligodeoxynucleotides and appropriately placed segmentsof modified oligodeoxy- or oligoribonucleotides. MBOs have segments ofphosphorothioate linkages and other segments of other modifiedoligonucleotides, such as methylphosphonate, which is non-ionic, andvery resistant to nucleases or 2′-O-alkyloligoribonucleotides. Methodsof preparing modified backbone and mixed backbone oligonucleotides areknown in the art.

Featured in this invention are connexin 43 antisense oligonucleotides orpolynucleotides comprising at least one unmodified nucleotide. In oneaspect, the connexin 43 antisense oligonucleotides or polynucleotidesmay comprise at least one modified nucleotide, and/or have at least onemodified internucleoside linkage, and/or at least one modified sugarmoiety. The modified internucleoside linkage may be, for example, aphosphorothioate linkage. In some aspects, for example, the connexin 43polynucleotide may comprise at least one nucleotide comprising aconformationally strained nucleotide, for example, a locked nucleic acid(LNA) or a bridged nucleic acid (BNA). The locked nucleotide may beselected, from one of the following types, for example: 2′-O—CH₂-4′(oxy-LNA), 2′-CH₂—CH₂-4′ (methylene-LNA), 2′-NH—CH₂-4′ (amino-LNA),2′-N(CH₃) CH₂-4′ (methylamino-LNA), 2′-S—CH₂-4′ (thio-LNA), and2′-Se—CH₂-4′ (seleno-LNA). In some aspects the modified nucleotide maybe a locked nucleic acid or an unlocked nucleic acid.

In some aspects of this invention, the connexin 43 antisenseoligonucleotide or polynucleotide has at least about 80%, 85%, 90%, 95%,97%, 98%, or 99% homology to a polynucleotide having a sequence selectedfrom SEQ ID NOs: 1 to 17 and 19.

Other Connexin 43 Modulators

Connexin modulators, for example, connexin 43 or 45 modulators,including peptides, peptidomimetics, antibodies, antibody fragments, andthe like, are also suitable modulators of gap junctions andhemichannels. Exemplary gap junction modulators may include, withoutlimitation, polypeptides (e.g. antibodies, binding fragments thereof,and synthetic constructs), and other gap junction blocking agents, andgap junction protein phosphorylating agents. In some aspects theconnexin modulator is a modulator of Cx26, Cx30, Cx31.1, Cx36, Cx37,Cx40, Cx43, Cx50, Cx57 or any other connexin in the eye or bloodvessels.

Connexin modulators, for example, connexin 43 or 45 modulators include,for example, monoclonal antibodies, polyclonal antibodies, antibodyfragments (including, for example, Fab, F(ab′)2 and Fv fragments; singlechain antibodies; single chain Fvs; and single chain binding moleculessuch as those comprising, for example, a binding domain, hinge, CH2 andCH3 domains, recombinant antibodies and antibody fragments which arecapable of binding an antigenic determinant (i.e., that portion of amolecule, generally referred to as an epitope) that makes contact with aparticular antibody or other binding molecule. These binding proteins,including antibodies, antibody fragments, and so on, may be chimeric orhumanized or otherwise made to be less immunogenic in the subject towhom they are to be administered, and may be synthesized, producedrecombinantly, or produced in expression libraries. Any binding moleculeknown in the art or later discovered is envisioned, such as thosereferenced herein and/or described in greater detail in the art. Forexample, binding proteins include not only antibodies, and the like, butalso ligands, receptors, peptidomimetics, or other binding fragments ormolecules (for example, produced by phage display) that bind to a target(e.g. connexin, hemichannel, or associated molecules).

Binding molecules will generally have a desired specificity, includingbut not limited to binding specificity, and desired affinity. Affinity,for example, may be a Ka of greater than or equal to about 10⁴ M-1,greater than or equal to about 10⁶ M-1, greater than or equal to about10⁷ M-1, greater than or equal to about 10⁸ M-1. Affinities of evengreater than about 10⁸ M-1 are suitable, such as affinities equal to orgreater than about 10⁹ M-1, about 101° M-1, about 10¹¹ M-1, and about10¹² M-1. Affinities of binding proteins according to the presentinvention can be readily determined using conventional techniques, forexample those described by Scatchard et al., (1949) Ann. N.Y. Acad. Sci.51: 660.

Exemplary compounds used for closing gap junctions (e.g. phosphorylatingconnexin 43 tyrosine and/or serine residue) have been reported in U.S.Pat. Nos. 7,153,822 and 7,250,397. Exemplary peptides andpeptidomimetics are reported in Green et al., WO2006134494. See alsoWO2006069181 and WO2003032964. Examples of other agents used for closinggap junctions include anti-connexin agents, for example anti-connexinpolynucleotides (for example, connexin inhibitors such as alpha-1connexin oligodeoxynucleotides), anti-connexin peptides (for example,antibodies and antibody binding fragments) and peptidomimetics (forexample, alpha-1 anti-connexin peptides or peptidomimetics), gapjunction closing or blocking compounds, hemichannel closing or blockingcompounds, and connexin carboxy-terminal polypeptides, e.g.,polypeptides that bind to ZO-1 or a ZO-1 binding site, anti-ZO-1polynucleotides.

By using data obtained from hydropathy plots, it has been proposed thata connexin contains four-transmembrane-spanning regions and two shortextra-cellular loops. The positioning of the first and secondextracellular regions of connexin was further characterized by thereported production of anti-peptide antibodies used forimmunolocalization of the corresponding epitopes on split gap junctions.Goodenough D. A. J Cell Biol 107: 1817-1824 (1988); Meyer R. A., J CellBiol 119: 179-189 (1992).

The extracellular domains of a hemichannel contributed by two adjacentcells “dock” with each other to form complete gap junction channels.Reagents that interfere with the interactions of these extracellulardomains can impair cell-to-cell communication. Peptide inhibitors of gapjunctions and hemichannels have been reported. See for example Berthoud,V. M. et al., Am J. Physiol. Lung Cell Mol. Physiol. 279: L619-L622(2000); Evans, W. H. and Boitano, S. Biochem. Soc. Trans. 29: 606-612,and De Vriese A. S., et al. Kidney Int. 61: 177-185 (2001). Shortpeptides corresponding to sequences within the extracellular loops ofconnexins were said to inhibit intercellular communication. Boitano S.and Evans W. Am J Physiol Lung Cell Mol Physiol 279: L623-L630 (2000).The use of peptides as inhibitors of cell-cell channel formationproduced by connexin (Cx) 32 expressed in paired Xenopus oocytes hasalso been reported. Dahl G, et al., Biophys J 67: 1816-1822 (1994).Berthoud, V. M. and Seul, K. H., summarized some of these results. AmJ., Physiol. Lung Cell Mol. Physiol. 279: L619-L622 (2000).

Anti-connexin agents include peptides having an amino acid sequence thatcomprises about 5 to 20 contiguous amino acids of a connexin proteinsuch as connexin 43 (SEQ. ID. NO:19), peptides having an amino acidsequence that comprises about 8 to 15 contiguous amino acids of connexin43, or peptides having an amino acid sequence that comprises about 11 to13 contiguous amino acids of connexin 43. Other anti-connexin agentsinclude a peptide having an amino acid sequence that comprises at leastabout 5, at least about 6, at least about 7, at least about 8, at leastabout 9, at least about 10, at least about 11, at least about 12, atleast about 13, at least about 14, at least about 15, at least about 20,at least about 25, or at least about 30 contiguous amino acids ofconnexin 43. Other anti-connexin 43 modulators comprise theextracellular domains of connexin 43, for example, peptide orpeptidomimetic comprising SRPTEKT (SEQ ID NO: 173) or VDCFLSRPTEKT (SEQID NO: 168). Other anti-connexin 43 modulators comprise the C-terminusregion of connexin 43, see WO2006/069181, or modified versions thereof.

In one aspect this invention relates to pharmaceutical compositions,articles of manufacture, and methods for treating neuropathic oculardisorders by administering a therapeutically effective amount of atleast one connexin or pannexin modulator to the eye of said subject. Insome aspects the neuropathic ocular disorder may be, for example, lossof retinal ganglion cells and/or glaucomatous optic neuropathy. In someaspects, administering a therapeutically effective amount of at leastone connexin modulator is effective for stopping, preventing, ortreating loss of retinal ganglion cells are further useful forincreasing the levels of neurotrophins in the glaucomatous optic nerveand decreasing vitreal glutamate concentrations.

In certain another aspect, gap junction modifying agent may include, forexample, compounds of Formula I or Formula II. In certain anotheraspect, gap junction modifying agent may include, for example, aliphaticalcohols; octanol; heptanol; anesthetics (e.g. halothane), ethrane,fluothane, propofol and thiopental; anandamide; arylaminobenzoate (FFA:flufenamic acid and similar derivatives that are lipophilic);carbenoxolone; Chalcone: (2′,5′-dihydroxychalcone); CHFs(Chlorohydroxyfuranones); CMCF(3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone); dexamethasone;doxorubicin (and other anthraquinone derivatives); eicosanoidthromboxane A(2) (TXA(2)) mimetics; NO (nitric oxide); Fatty acids (e.g.arachidonic acid, oleic acid and lipoxygenase metabolites; Fenamates(flufenamic (FFA), niflumic (NFA) and meclofenamic acids (MFA));Genistein; glycyrrhetinic acid (GA): 18a-glycyrrhetinic acid and18-beta-glycyrrhetinic acid, and derivatives thereof; lindane;lysophosphatidic acid; mefloquine; menadione;2-Methyl-1,4-naphthoquinone, vitamin K(3); nafenopin; okadaic acid;oleamide; oleic acid; PH, gating by intracellular acidification; e.g.,acidifying agents; polyunsaturated fatty acids; fatty acid GJICinhibitors (e.g., oleic and arachidonic acids); quinidine; quinine; alltrans-retinoic acid; and tamoxifen.

Polynucleotide Homologues

Homology and homologues are discussed herein (for example, thepolynucleotide may be a homologue of a complement to a sequence inpannexin mRNA). Such a polynucleotide typically has at least about 70%homology, preferably at least about 80%, at least about 90%, at leastabout 95%, at least about 97% or at least about 99% homology with therelevant sequence, for example, over a region of at least about 15, atleast about 20, at least about 25, at least about 30, at least about 40,at least about 50, or at least about 100 more contiguous nucleotides (ofthe homologous sequence).

Homology or sequence identity may be calculated based on any method inthe art. For example, the UWGCG Package provides the BESTFIT programthat can be used to calculate homology (Devereux, et al. (1984) NucleicAcids Research 12, p 387-395). The PILEUP and BLAST algorithms can alsobe used to calculate sequence identity or align sequences, for example,as described in Altschul, S. F. (1993), J Mol Evol 36: 290-300;Altschul, et al (1990), J Mol Biol 215: 403-10. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). The BLASTalgorithm performs a statistical analysis of the similarity between twosequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci.USA 90: 5873-5787. The homologous sequence typically differs from therelevant sequence by at least about (or by no more than about) 2, 5, 10,15, 20, or more nucleotide differences (which may be substitutions,deletions, or insertions). These differences can be measured across anyof the regions mentioned above in relation to calculating sequenceidentity or homology.

The homologous sequence typically hybridizes selectively to the originalsequence at a level significantly above background. Selectivehybridization is typically achieved using conditions of medium to highstringency (for example 0.03M sodium chloride and 0.03M sodium citrateat from about 50° C. to about 60° C.). However, such hybridization maybe carried out under any suitable conditions known in the art (seeSambrook, et al. (1989), Molecular Cloning: A Laboratory Manual). Forexample, if high stringency is required, suitable conditions include0.2×SSC at 60° C. If lower stringency is required, suitable conditionsinclude 2×SSC at 60° C.

Peptide and Polypeptide Anti-Pannexin Agents

Pannexin binding proteins, including peptides, peptidomimetics,antibodies, antigen-binding antibody fragments, and the like, are alsosuitable modulators of adherens junctions.

Binding proteins include, for example, monoclonal antibodies, polyclonalantibodies, antibody fragments (including, for example, Fab, F(ab′)2 andFv fragments; single chain antibodies; single chain Fvs; and singlechain binding molecules such as those comprising, for example, a bindingdomain, hinge, CH2 and CH3 domains, recombinant antibodies, and antibodyfragments which are capable of binding an antigenic determinant (i.e.,that portion of a molecule, generally referred to as an epitope) thatmakes contact with a particular antibody or other binding molecule.These binding proteins, including antibodies, anti-binding antibodyfragments, and so on, may be chimeric or humanized or otherwise made tobe less immunogenic in the subject to whom they are to be administered,and may be synthesized, produced recombinantly, or produced inexpression libraries. Any binding molecule known in the art or laterdiscovered is envisioned, such as those referenced herein and/ordescribed in greater detail in the art. For example, binding proteinsinclude not only antibodies, and the like, but also ligands, receptors,peptidomimetics, or other binding fragments or molecules (for example,produced by phage display) that bind to a target (e.g., a pannexinprotein).

Binding molecules will generally have a desired specificity, includingbut not limited to binding specificity, and desired affinity. Affinity,for example, may be a K_(a) of greater than or equal to about 10⁴ M⁻¹,greater than or equal to about 10⁶M⁻¹, greater than or equal to about10⁷ M⁻¹, greater than or equal to about 10⁸ M. Affinities of evengreater than about 10⁸ M⁻¹ are suitable, such as affinities equal to orgreater than about 10⁹ M⁻¹, about 10¹⁰ M⁻¹, about 10¹¹ M⁻¹, and about10¹²M⁻¹. Affinities of binding proteins according to the presentinvention can be readily determined using conventional techniques, forexample those described by Scatchard, et al., 1949 Ann. N.Y. Acad. Sci.51: 660.

Anti-pannexin agents include peptides comprising an amino acid sequencecorresponding to a pannexin domain motif from a pannexin protein (e.g.,pannexin1, pannexin2, pannexin3 etc.). Other embodiments are directed toan anti-connexin agent that is a peptide having an amino acid sequencethat comprises at least about 5, at least about 6, at least about 7, atleast about 8, at least about 9, at least about 10, at least about 11,at least about 12, at least about 13, at least about 14, at least about15, at least about 20, at least about 25, or at least about 30contiguous amino acids encoded by a pannexin gene, for example, anpannexin1 gene as set forth in Example 1, below. In certainanti-connexin agents provided herein, the extracellular domains ofpannexin1 may be used to develop the particular peptide sequences. Thepeptides need not have an amino acid sequence identical to thoseportions of naturally occurring pannexin, and conservative amino acidchanges may be made such that the peptides retain binding activity orfunctional activity. Alternatively, peptides may target other regions ofthe extracellular domain.

Anti-pannexin peptides may comprise sequences corresponding to a portionof a pannexin extracellular domain with conservative amino acidsubstitutions such that peptides are functionally active anti-pannexinagents. Exemplary conservative amino acid substitutions include forexample the substitution of a nonpolar amino acid with another nonpolaramino acid, the substitution of an aromatic amino acid with anotheraromatic amino acid, the substitution of an aliphatic amino acid withanother aliphatic amino acid, the substitution of a polar amino acidwith another polar amino acid, the substitution of an acidic amino acidwith another acidic amino acid, the substitution of a basic amino acidwith another basic amino acid, and the substitution of an ionizableamino acid with another ionizable amino acid.

Manufacture and Stability

The polynucleotides of this invention can be manufactured usingsolid-phase chemistries for synthesizing oligonucleotides. In oneaspect, the formulations of this invention will comprise a salt of thepolynucleotides of this invention, such as the sodium salt of thepolynucleotides of this invention. In one embodiment the formulation maycomprise the sodium salt of a polynucleotide having any one of SEQ. ID.NO:1-16 or a portion of SEQ ID NO:17, for example. In some embodiments,the polynucleotide having any one of SEQ. ID. NO:1-16 may be a modifiedoligodeoxynucleotide having any one of SEQ. ID. NO:1-16, or a portion ofSEQ ID NO:17.

In some embodiments, the formulations of this invention aresubstantially pure. By substantially pure is meant that the formulationscomprise less than about 10%, 5%, or 1%, and preferably less than about0.1%, of any nucleotide or non-nucleotide impurity. In some embodimentsthe total impurities, including metabolities of the connexin 43modulating agent, will be not more than 15%. In some embodiments thetotal impurities, including metabolities of the connexin 43 modulatingagent, will be not more than 12%. In some embodiments the totalimpurities, including metabolities of the connexin 43 modulating agent,will be not more than 11%. In other embodiments the total impurities,including metabolities of the connexin 43 modulating agent, will be notmore than 10%.

In some embodiments, the purity of the formulations of this inventionmay be measured using a method selected from anion exchange HPLC(AEX-HPLC) or mass spectrometry. Mass spectrometry may include LC/MS, orLC/MS/MS. The assay may in some embodiments comprise both AEX-HPLC andLC/MS.

Sterile compositions comprising the connexin 43 modulating agents ofthis invention prepared using aseptic processing by dissolving theanti-connexin modulating agent in the formulation vehicle. In oneembodiment, the formulation may also be sterilized by filtration.Excipients used in the manufacture of the formulations of this inventionare widely used in pharmaceutical products and released to pharmacopeialstandards.

EXAMPLES

Various small organic molecules have been reported to have activity ininhibition of gap junction or hemichannel currents. They includetriarylmethanes (TRAMS), quinine, mefloquine, fenamates,2-aminophenoxyborate and derivatives, glycyrrhetinic acid andderivatives, volatile anesthetics such as halothane and ethane,lipophilic compounds such as long-chain alcohols (e.g., heptanol andoctanol), fatty acid amides including oleamide, cyclodextrins,cisplatin, polyamines and tetraalylammonium ions. An increasing numberof studies have also reported on the inhibition of gap junction channelsand hemichannels using peptides corresponding to specific sequenceswithin extracellular loops E1 and E2 involving the conserved QPG andSHVR (SEQ ID NO: 18) motifs of E1 (Gap26 peptide) and the SRPTEK (SEQ IDNO: 20) motif in E2 (Gap27 peptide) as well as the cytoplasmic loop(Gap19 peptide). The most potent such peptidomimetic is Peptide5(VDCFLSRPTEKT (SEQ ID NO: 168)).

Peptide5 is an established gap junction channel blocker that can operatein a dose dependent manner, with lower doses blocking gap junctionhemichannel opening and higher doses uncoupling gap junctions betweencells. See, e.g., O'Carroll et al, 2008. With sustained low doseapplication of Peptide5 there is also gradual loss of gap junctioncoupling, considered to be peptide interference with hemichannel docking(in parallel with gradual removal of existing gap junctions duringnormal turnover). Peptide5 has proven to be effective in a number of invitro, ex vivo and in vivo (animal) studies, especially when used atdoses that block hemichannels without uncoupling gap junctions (see forexample Davidson et al, 2012; Danesh-Meyer et al, 2012; O'Carroll et al,2013). The results in O'Carroll et al, 2008 indicate that Peptide5 atlow or high concentration blocks hemichannels, but will uncouple gapjunctions directly at high concentrations. Peptide5 data is shown herefor comparison with tonabersat.

Example 1: Methods and Materials for hCMVEC and ARPE-19 Cell Studies

In the below Examples 2, 4, 5, 8 and 10 using human cerebralmicrovascular endothelial cells (hCMVEC), cells were prepared by growingthem in collagen coated (30 μg/mL) T25 or T75 flasks maintained inMedium M199 supplemented with 10% FCS, 1 μg/mL hydrocortisone, 3 ng/mLhFGF, 1 ng/mL hEGF, 10 μg/mL heparin, 1× penicillin streptomycinneomycin (Life Technologies), and cAMP (100 μL 100% cAMP to 100 mLmedia). Cells were maintained at 37° C. with 95% 02 and 5% CO₂.

In the below Examples 3, 6 and 7 using human retinal epithelial(ARPE-19) cells, the cells were grown in DMEM/F: 12 (Invitrogen),supplemented with 10% fetal calf serum (FCS), 100 units/mL ofpenicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of Fungizone®Antimycotic (Invitrogen).

With respect to the chemicals used in these studies, tonabersat(Medchemexpress, USA) was dissolved in dimethyl sulfoxide (DMSO) at astock concentration of 100 mM. Ammonium chloride (NH₄Cl, Sigma) wasdissolved in dH20 at a stock concentration of 100 mM. Connexin43 mimeticpeptide (Peptide5, sequence VDCFLSRPTEKT (SEQ. ID NO:168)) (Auspep,Australia), and Peptide 8 (CDEQSAFRCNTQQ (SEQ. ID NO:241)) weresynthesized at purity >95% and dissolved in dH20 at a concentration of10 mM. Probenecid (C13H19NO4S, Sigma) was solubilized in 1 M NaOH at aconcentration of 50 mg/mL.

All animal model procedures were conducted in compliance with the ARVOStatement of Use of Animals in Ophthalmic and Vision Research and wereapproved by the Animal Ethics Committee of the University of Auckland.Seventeen adult male Wistar rats weighing between 240-260 g wereobtained from the Vernon Jenson Unit of the University of Auckland andhoused with a 12-hour light/dark cycle and received food and water adlibitum.

Example 2: Scrape Load Assay

In this Example, human cerebral microvascular endothelial cells (hCMVEC)were grown in collagen coated (1 μg/mL) T25 flasks maintained in MediumM199 supplemented with 10% FBS, 1 μg/mL hydrocortisone, 3 ng/mL hFGF, 1ng/mL hEGF, 10 μg/mL heparin and cAMP (100 μL 100% cAMP to 100 mL media)in a humidified atmosphere at 37° C. with 5% CO2. One day prior toexperimentation, these cells were trypsinized and plated at a density of4×105 cells/well in a collagen-coated 12 well-plate in the same medium.For treatments, cells were cultured in the presence of peptide5, acontrol gap junction channel blocker (carbenoxolone), or tonabersat forthe respective time intervals prior to scrape dye loading. The blockerswere also present in the respective scrape loading solutions.

For scrape loading the hCMVEC cells were washed three times withphosphate buffered saline (PBS) (without divalent cations). A solutionof PBS+0.05% lucifer yellow was added to cells. The cells then receiveda scrape to the monolayer using a size 10 carbon steel surgical bladeand incubated in the dark at 37° C. with 5% CO2 for 5 minutes. The cellswere washed with PBS four times and then 1 mL of PBS added forfluorescent microscopy.

The live cells were then imaged on a Nikon TE2000E fluorescentmicroscope whilst maintained in a chamber heated to 37° C. with 5% CO2.Three images pairs were obtained at 10× magnification for each of aphase contrast image to view total cells and a fluorescent image tovisualize Lucifer yellow using 488 nm excitation and a GFP filter block.Gap junctional communication was determined by counting the number ofLucifer yellow positive cells and presented as a percentage to thepositive cells of the control group. Data was graphed as mean+standarderror of the mean using Prism v5.02 software. Significance wasdetermined through students two-tailed, unpaired, T Tests. *=p<0.05,**=p<0.001, ***=p<0.001.

Results in FIG. 1 (using peptide5 as an example) show that dye taken upby cells adjacent to the scrape spreads to neighboring cells. Peptide5at 100 μM concentration added immediately before scraping has littleeffect on cell-cell transfer of dye through gap junctions. The higher500 μM concentration of peptide5 lead to a marked reduction in dyetransfer. With longer, 2-hour incubations, dye transfer is reduced withpeptide5 at both concentrations. This indicates that peptide5 can beused in a dose dependent manner given the finding that it can block gapjunctions immediately at higher concentrations and that, with sustainedtreatment, low dose application leads to a reduction in gap junctioncoupling, the latter being considered to be peptide interference withhemichannel docking (in parallel with gradual removal of existing gapjunctions during normal turnover). The graph to the right of FIG. 3compares the efficacy of peptide5 block (high dose) with thenon-specific gap junction channel blocker carbenoxolone.

FIG. 3 shows comparable scrape loading dye transfer for tonabersat at 50μM concentration. Added immediately tonabersat has a statisticallysignificant gap junction uncoupling effect within 1 minute, but blockincreases in a time dependent manner indicating that tonabersat is alsoacting in a similar manner to peptide5. Without wishing to be bound byany particular theory, the inventors consider this may be the result ofinterference with hemichannel docking or triggering internalization ofhemichannels in parallel with gradual removal of existing gap junctionsduring normal turnover, internalisation of gap junctions (see FIG. 4).

Example 3: Immunohistochemistry of ARPE-19 Retinal Epithelial CellsTreated with Peptide5 and Tonabersat

Cells were seeded at 0.05×106 per well in 8-well sterile glassmicroscope chamber slides (Falcon, Corning Life Sciences), and themonolayer was labeled for connexin expression at −90-100% confluency.The following steps were conducted at room temperature. Cells were fixedin 4% paraformaldehyde (P6148, Sigma-Aldrich) in PBS with 0.1 mM CaCl2for 10 min, and washed three times in PBS with 0.1 mM CaCl2. Afterblocking in 10% normal goat serum in PBS with 0.1 mM CaCl2 for one hour,cells were incubated overnight in primary antibody solution (rabbitanti-Cx43, Sigma C6219) diluted in PBS with 0.1 mM CaCl2. The chamberslides were then washed three times with PBS containing 0.1 mM CaCl2 for10 minutes, and incubated for a further 45 minutes in goat anti-rabbitAlexa 568 conjugated secondary antibody (Invitrogen #A11036). Cells werecounter labeled with DAPI for 5 minutes and washed three times with PBScontaining 0.1 mM CaCl2. Chamber wells were removed and cells weremounted anti-fade medium (Citiflour AF-1). Dilution ratios for specifiedprimary and secondary antibodies were used according to manufacturer'srecommendations.

FIG. 4 shows DAPI (nuclear staining) and labelling of the gap junctionprotein connexin43 (left panels) and matching connexin43 only labelling(right hand panels). In the untreated cells gap junction labelling isprimarily at cell-cell interfaces (top panels). In the lower panelsshowing cells treated for one hour with peptide5 (500 μM) the cell-cellinterfaces are still apparent but the bulk of the gap junctions havebeen internalized and label is cytoplasmic. This indicates that once thepeptide uncouples gap junctions, it triggers internalization andjunction degradation. FIG. 3 shows an equivalent experiment withuntreated and tonabersat treated cells (50 μM, 6 hours). Tonabersattreatment over time results in total gap junction loss at cell-cellinterfaces.

Thus, which the scrape-loading experiments in Example 2 showed thefunctional loss of gap junctions, this Example 3 explains how this canhappen, i.e., by the loss of gap junctions between cells.

Example 4: Electrophysiology of Gap Junction Hemichannels

Whole-cell voltage clamp studies were conducted on connexin deficientHeLa cells, Connexin43 transfected HeLa cells and isolated humancerebral microvascular endothelial cells (hCMVEC).

HeLa cells were seeded at 0.045×106 cells/mL on 18 mm rat collagen Icoated coverslips 16-24 hours before each experiment. Cx43IRESeGFP HeLacells were cultured overnight in DMEM supplemented with 10% FCS and 1μg/mL Puromycin at 37° C. at 95% O2 and 5% CO2. Control Cx-deficientcells were maintained in DMEM supplemented with 10% FCS. Coverslips weretransferred to a chamber continuously perfused with modifiedKrebs-Ringer solution containing (in mM): 140 NaCl, 5.4 KCl, 1 MgCl2, 10mM Hepes and pH adjusted to 7.4 at room temperature. The internalpipette solution contained (in mM): 130 KCl, 10 sodium aspartate, 0.26CaCl2, 2 EGTA, 5 tetraethylammonium-C1, 1 MgCl, 3 MgATP, and 5 Hepes atpH 7.2; and was kept on ice. The solutions are described in Contreras JE, et al., Cell Communication and Adhesion (2003), 10:245-249. Toresolve single currents in whole-cell configuration, cells with lowcapacitance (<10 pF) and high resistance pipettes (6-10 mOhm) with aseal resistance of (>5 GOhm) were selected for recording and analysis.

HCMVEC Cells were placed into an extracellular artificial cerebralspinal fluid solution containing (in mM): 144 NaCl, 3.3 KCl, 1.8 CaCl2,1.8 MgCl2, 10 mM glucose, pH: 7.385 at temperature 37° C. Theintracellular pipette solution contained (in mM): 130 KCl, 10 sodiumaspartate, 0.26 CaCl2, 2 EGTA, 5 tetraethylammonium-C1, 1 MgCl2,3 MgATP,5 Hepes at pH 7.2; and was kept on ice to prevent MgATP breakdown.Pipette resistance was 3.4MΩ with seal resistance=1.3GΩ. Voltage andperfusion protocols are conducted simultaneously, with cellscontinuously perfused. Recordings for analysis were taken beforeTonabersat perfusion, during and after washout. For channel blockstudies, 50 μM Tonabersat was washed in for three mins prior torecording, and subsequently washed out three mins before taking thewashout recordings.

FIG. 5 shows recordings in connexin43 transfected HeLa cells (toptraces) and connexin null HeLa cells (bottom traces). As the voltagesteps are raised, increased channel activity is observed in thetransfected cells which is not present in the connexin null cells. Thischannel activity in the transfected cells is therefore the result ofconnexin43 hemichannel opening. FIG. 6 shows traces fromconnexin43-transfected HeLa cells that have been treated with thenon-specific channel blockers carbenoxolone and LaC13, and with peptide5(100 μM). The non-specific channel blockers show virtually completehemichannel block with little channel activity remaining. Peptide5 alsoshows significant hemichannel blockade.

FIG. 7 shows an equivalent experiment in HCMVEC cells prior to additionof tonabersat (50 μM), during incubation with tonabersat, and afterthree minutes of washout. Tonabersat almost completely abolisheshemichannel activity, with some activity returning after washout. Theseresults indicate that tonabersat is a very effective hemichannelblocker, that may exceed the efficacy of peptide5 at the concentrationsused, and that active hemichannels are recoverable.

Discussion

The inventors have surprisingly determined that the establishedconnexin43 channel blocker peptide5 and tonabersat exhibit a similarmode of action. At high concentrations peptide5 can block connexin43hemichannels and can uncouple connexin43 gap junctions. At lower dosespeptide5 blocks connexin43 hemichannels but does not have an immediateeffect on gap junction coupling. With sustained low dose application ofpeptide5 there is also gradual loss of gap junctions at the cell-cellinterface, indicating peptide interference with hemichannel docking (inparallel with gradual removal of existing gap junctions during normalturnover). The results herein indicate that tonabersat is primarily ahemichannel blocker, and is also able to block gap junction channelsand, like peptide5, can be used to decrease gap junction coupling,possibly by preventing hemichannel docking to form replacement gapjunction channels during turnover or by triggering internalization fromthe plasma membrane. In these experiments, tonabersat was shown to blockconnexin hemichannels within one minute and provide a decrease incell-cell coupling within one minute.

Tonabersat can reduce connexin26 expression in the central nervoussystem through a specific receptor and the p38 pathway. The inventorshave surprisingly discovered that tonabersat can be used to blockhemichannels in a number of cell types (hCMVEC, HeLa, ARPE), includingendothelial cells. Thus, the inventors have surprisingly discovered thattonabersat is not central nervous system specific. The results alsoindicate that tonabersat has significant efficacy in blocking conenxin43hemichannels, and that it can be used to uncouple connexin43 gapjunctions. Thus, it is also not connexin isoform specific.

The results support the inventor's observation that tonabersat isprimarily a connexin hemichannel blocker, but can be used at higherdoses to uncouple gap junction. Its hemichannel block efficacy and speedof action, and the fact that it could be washed out within three minutesto restore channel activity, implies it has a direct effect onhemichannels, as opposed to a slower pathway whereby mRNA or proteinexpression is affected.

Example 5: Connexin43 Hemichannels and Pannexin Channels Release ATPDuring Ischaemia Injury-Reperfusion In Vitro

Using an in vitro ischemia injury model, the Cx43 hemichannel andpannexin channel mediated ATP release was differentiated using theconnexin hemichannel blocking mimetic peptide5 at 100 μM concentration(S. J. O'Carroll, et al., Cell communication & adhesion 15, 27 (May,2008)) and 1 mM Probenecid (W. R. Silverman et al., The Journal ofbiological chemistry 284, 18143 (Jul. 3, 2009)).

In Vitro Ischaemia Injury-Reperfusion ATP Assay

Human cerebral microvascular endothelial cells (hCMVEC) were trypsinised(TrypLE Express, Life technologies) and plated at a density of 0.025×106cells per well in a collagen-coated 12-well plate (1 μg/cm2) a day priorto the experiment. Cells were incubated in culture medium overnight.Hypoxic, acidic, ion-shifted Ringer injury solution that mimics ionicconcentrations and acid-base shifts of the interstitial space inhypoxic-ischemic brains (A. Bondarenko, et al., Glia 34, 143 (Apr. 15,2001)) was used to trigger hemichannel opening. The injury solution (inmM): 38 NaCl, 13 NaHCO3, 3 Na-gluconate, 65 K-gluconate, 38 NMDG-Cl, 1NaH2PO4, 1.5 MgCl2, was bubbled in N2 gas (>99% purity, 20 L/min) for 5minutes and pH adjusted to 6.6 using 5 M HCl. Control solution thatmimics physiological conditions contained (in mM): 124 NaCl, 3 KCl, 26NaHCO3, 26 NaHCO3, 1 NaH2PO4, 1.3 CaCl2, 1.5 MgCl2, and 10 Glucose, andpH 7.4 was adjusted using 5 M HCl (Bondarenko, 2001). In the injurymodel, hCMVEC cells were incubated in 500 μL control solution, or injurysolution, or Tonabersat (0.1-50 μM)+/−Probenecid (1 mM) dissolved ininjury solution for 2 hours in 37° C. in 95% 02 and 5% CO2. Following a2-hour injury the solutions were immediately placed on ice. In theinjury and reperfusion model, hCMVEC cells were incubated in 500 μL ofnormal solution or injury solution for 2 hours at 37° C. in 95% 02 and5% CO2. Following 2 hour injury, the solutions were discarded andreplaced with 500 μL of the normal solution, or Tonabersat (10 μM)dissolved in normal solution, and were incubated for 2 hours at 37° C.in 95% 02 and 5% CO2. These solutions were also then immediately placedon ice. Peptide5 (100 μM)+/− Probenecid (1 mM) were used as controls forboth injury and injury-reperfusion models. ATP concentration in thesamples was determined using a Luciferin/luciferase bioluminescencereaction (ATP Determination Kit, Molecular Probes) and detected using aluminescence plate reader (VICTOR X, Perkin Elmer #2030-0010). Standardcurves were generated in each experiment from an ATP standard (500 nM)to quantify the concentration of ATP in the test samples. The data ispresented as mean±standard error relative to the injury orinjury-reperfusion control. Statistically significant differencesbetween samples were identified using one-way analysis of variance andTukey's multiple comparisons test.

FIG. 8 summarizes the total ATP released from a sub-confluent culture ofhuman cerebral microvascular endothelial cells (hCMVEC) following twohours in the in vitro injury model. When compared with injury group(100±2.2%), there was a basal level of 35±2.6% ATP released from noinjury control that may reflect normal cellular activity and/or deadcells prior to injury. A significant difference in extracellular ATP wasobserved between no injury and injury control (p<0.0001) (FIG. 10A).Compared to injury (100±2.2%), Peptide5 significantly lowered ATP downto 73.9±2.1% (p<0.0001), and Probenecid also significantly reduced ATPdown to 83.1±4.5% (p<0.001) (FIG. 10A). Interestingly, a combinedtreatment of Peptide5 and Probenecid reduced ATP to a level that wascomparable to 100 μM carbenoxolone (CBX), a non-specific blocker thatblocks both Cx43 hemichannels and pannexin channel, with no significantdifference observed between these two groups (p=0.28). Data from theseexperiments demonstrate that both connexin hemichannels and pannexinchannels open under ischemic conditions.

Inhibition of Cx43 Hemichannels by Tonabersat is ConcentrationDependent, but Only in the Absence of Pannexin Channel Activity DuringInjury In Vitro

The following experiment was performed to demonstrate that Cx43HC-mediated ATP release from injured hCMVEC cells decreases as theconcentration of tonabersat increases. Utilizing the injury model inFIG. 8A, the effect of Tonabersat on Cx43 HC mediated ATP release wasisolated using Probenecid to inhibit Panx channels. Compared to injury(100±1.2%), ATP release was significantly reduced at Tonabersatconcentrations of 0.1 μM (85.9±2.4%), 1 μM (75.9±2.3%), 10 μM(58.5±1.9%), and 100 μM (70.5±3.9%) in combination with 1 mM Probenecid(FIG. 10B). Increasing the concentration of Tonabersat (0.1 μM to 10 μM)significantly reduced Cx43 HC-mediated ATP release (FIG. 8B). Maximalinhibition was achieved at 10 μM tonabersat, and this was significantlygreater by 12.1±3.2% than the highest concentration of 100 μM (p=0.005)(FIG. 8B). However, there remained a significant difference of 14.9±4%between CBX, and 10 μM Tonabersat in 1 mM Probenecid (p=0.0059) (FIG.10B). Tonabersat (0.1 μM to 100 μM) significantly reduced Cx43 HCmediated ATP release compared to injury when in combination withProbenecid; however, Tonabersat did not reduce ATP release from hCMVECcells in the absence of 1 mM Probenecid (p>0.09) (FIG. 8C).

Connexin43 Hemichannels, but not Pannexin Channels, Release ATP DuringReperfusion In Vitro

The role of Cx43 hemichannel and pannexin channel during the reperfusionperiod post-ischemia (return to normal media post two hours in HAIRsolution) was further characterized. A significant difference in ATPrelease between no injury-reperfusion (No IR) and injury-reperfusioncontrol (IR) (p=0.0006) was observed (FIG. 11). While Peptide5significantly lowered ATP release to 70.3±4.4% (p=0.0196), Probeneciddid not significantly reduce ATP compared to reperfusion injury(99.3±4.4%, p>0.99) (FIG. 9). A combined treatment of peptide5 andprobenecid also significantly reduced ATP compared to reperfusion injury(71.1±7.4%, p=0.025), but this was comparable to Peptide5 treatmentalone (FIG. 11). In contrast to injury only (FIG. 10B), CBXsignificantly lowered ATP release further to 33.8±33.5% compared toPeptide5 only (p=0.0127) (FIG. 9).

A Low Concentration of Tonabersat Inhibits Cx43 Hemichannel Mediated ATPRelease During Reperfusion In Vitro

The ischaemia-reperfusion assay demonstrated that Probenecid had nosignificant effect on the release of ATP from hCMVEC cells duringreperfusion (FIG. 9). The low dose at 10 μM Tonabersat alone will besufficient to inhibit the ATP released from hCMVEC cells in vitro, andthe level of inhibition will be comparable to Peptide5. A significantreduction in ATP in the presence of 10 μM Tonabersat was observed(68.9±6.9%, p=0.02) which was comparable to 100 μM Peptide5 duringreperfusion injury in vitro (FIG. 9).

Example 6: High Concentrations of Tonabersat Causes Internalization ofCx43 Plaques in ARPE-19 Cells Immunocytochemistry and Quantification ofCx43 Plaques

ARPE-19 cells were grown until confluent in 8-well glass chamber-slides(BD Falcon). Confluent monolayers of ARPE-19 cells were incubated at afinal concentration of 5 to 500 μM) Tonabersat, and/or 10 mM NH4Cl (H.Qin, et al., The Journal of biological chemistry 278, 30005 (Aug. 8,2003)) in culture medium for 1 or 6 hours. Cells were fixed in 4% PFA(ProSciTech) at pH 7.4 for 10 min at room temperature, permeabilizedwith 0.05% Triton-X100 in PBS, and incubated in 10% normal goat serum toblock non-specific labelling. Cells were rinsed three times with PBScontaining 0.1 mM CaCl2 between each fixation, permeabilization, andblocking steps. Cx43 polyclonal rabbit antibody (C6219, Sigma, 1:2000)was applied for 24 hours, followed by a goat anti-rabbit Alexa Fluor®568 secondary antibody (Invitrogen, 1:200) for 45 minutes. Nuclei werecounterstained with DAPI (Invitrogen) at 10,000-fold dilution for 5mins, and mounted with Citifluor™ mounting medium. Images werevisualised and captured using a 63× oil immersion lens on an OlympusFV1000 upright confocal laser scanning microscope and software. Thetransfluor feature in MetaXpress® Image acquisition and analysissoftware (Version 5.3.0.1, Molecular Devices) was used to automate andquantify the total area of Cx43 plaques per image. Results representmean±standard error and statistical tests were conducted using one-wayANOVA and Tukey's multiple comparisons test.

Immunocytochemistry, as described above, was used to visualise thedistribution and size of the Cx43 GJ plaques in a native human cellline, retinal pigmented epithelial cells (ARPE-19), which reportedly hasan abundant expression of Cx43 (C. M. Hutnik, et al., Investigativeophthalmology & visual science 49, 800 (February, 2008)). Cx43 plaquesbetween cell-to-cell contacts were observed that were labelled in aregular tile-like pattern under basal conditions (FIG. 13A). Cx43labelling was also visible in the perinuclear region, which indicated anintracellular pool of Cx43 in the Golgi apparatus (J. Das Sarma et al.,Journal of cell science 114, 4013 (November, 2001)). However,significant reductions in Cx43 plaques were observed following 1 hourexposure to Tonabersat at concentrations of 100 μM (52.8±5.6%,p=0.0012), 200 μM (63.2±5.6%, p=0.0286), and 500 μM (63.2±5.6%,p=0.0420) compared to normal control (FIG. 13B). The reductions in Cx43plaques were visible between cell-to-cell contacts and the intracellularpool (FIG. 10A, top image). However, compared to control, there was nosignificant reduction in Cx43 plaques at lower concentrations of 5 μM(p=0.99), 10 μM (p=0.5777), or 50 μM (p=0.1146) (FIG. 11B).

The results show that tonabersat causes internalization of Cx43 GJs fromthe plasma membrane. The reductions in Cx43 plaques were demonstrated tobe time-dependent by exposing ARPE-19 cells in Tonabersat (50 to 500 μM)for an extended period of 6 hours. Comparable to FIG. 10, 6 hourtreatment in tonabersat also led to reductions in Cx43 labeling betweencell-to-cell contacts and the intracellular pool (FIG. 12A).Furthermore, the total area of Cx43 plaques was also significantlyreduced at 100 μM (66.5±5.4%, p<0.0001), 200 μM (62.5±2.3%, p<0.0001),and 500 μM (60.2±13.6%, p<0.0001).

Tonabersat Targets Internalized Cx43 Plaques for Degradation Via theLysosomal Pathway

In contrast to peptide 5, which only internalized gap junctions, it wasalso demonstrated that tonabersat facilitates turnover of Cx43 plaquesfrom the plasma membrane. See FIG. 11. The lysosomal degradation pathwayin ARPE-19 cells was immobilized using ammonium chloride (NH4C1), aweak-base lysosome inhibitor (H. Qin, et al., The Journal of biologicalchemistry 278, 30005 (Aug. 8, 2003)). Following 1 hour treatment inTonabersat and 10 mM NH4Cl, Cx43 plaques were removed from cell-to-cellcontacts, but remained in the cytoplasm with punctate labellingexpressed along the intracellular region immediately below the plasmamembrane. In addition there was now dense labelling visible around theperinucleus (in what appears to be the golgi body) (FIG. 14A, lowerimage). In tonabersat with NH4Cl, significantly more Cx43 labelling waspresent (up 104.8%) compared to 100 μM Tonabersat alone (p<0.001), andup 65.4% more when compared to 200 μM Tonabersat alone (p=0.0002) (FIG.10C).

Following 6-hour treatment in tonabersat and 10 mM NH4Cl, punctate Cx43labeling was distributed throughout the cell (FIG. 15A, lower image). Incontrast to the 1-hour period, there were no visible Cx43 labelingbetween cell-to-cell contacts following treatment in Tonabersat andNH4Cl (FIG. 15A, lower image). However, NH4Cl significantly increasedthe amount of Cx43 labeling (total area), up 67.9% 5 in 50 μM(p=0.0015), up 80.3% in 100 μM (p=0.0001), and up 87.9% in 200 μM(p=0.0062) Tonabersat when compared to Tonabersat treatment alone (FIG.12C). There were no significant differences in the total area of Cx43plaques per cell between untreated, DMSO (vehicle control), or NH4Clonly control following 1-hour and 6-hour post incubation (FIGS. 10B and12B).

Example 7: Tonabersat does not Affect Cx43 mRNA Transcription orTranslation

Tonabersat does not affect Cx43 mRNA transcription or translation, asindicated in published literature. Rather, the observedtonabersat-mediated down-regulation of Cx43 is caused by activation ofinternalization and degradation pathways.

Real-Time Reverse Transcription PCR

Real time reverse transcription PCR was used to determine the relativelevel of Cx43 mRNA. A confluent monolayer of ARPE-19 cells was treatedwith Tonabersat for 1 hour. Cells were harvested with TRIzol® (LifeTechnologies), and total RNA was isolated using SuperScript III FirstStrand kit (Invitrogen) according to manufacturer's instructions. AllRNA samples were quantified using a nanodrop 1000 micro volumespectrophotometer (Thermo Scientific). cDNA from samples weresynthesised using SuperScript® III First-Strand Synthesis SuperMix(Invitrogen) according to manufacturer's instructions. Primer sequencesfor hCx43, GenBank Accession No. M65188.1, were obtained from theHarvard Primer bank. The sequences for human Cx43 were forward5′-TGGTAAGGTGAAAATGCGAGG-3′ (SEQ. ID NO:242) and reverse5′-GCACTCAAGCTGAATCCATAGAT-3′ (SEQ. ID NO:243). The appropriatereference gene to be used for relative fold-difference in Cx43 mRNA wasdetermined by screening commonly used house-keeping genes, ß-actin[ACTB], Hypoxanthine phosphoribosyltransferase 1 [HPRT1], peptidylprolylisomerase A [PPIA], and glyceraldehyde-3-phosphate dehydrogenase[GAPDH]. We chose beta-actin as the control gene as it showed nosignificant difference in expression between the treatment groups. AllPCR reactions were performed on the Rotor gene600 (Qiagen) using a cycleprogram of 50° C. for 2 min then 95° C. for 2 min; 95° C. for 15 s and60° C. repeated 40 times; 95° C. for 15 s, 60° C. for 15 s, 95° C. for15 s, in order to generate threshold cycles for relative quantification(Ct). Relative changes in Cx43 mRNA expression were calculated asfold-differences against 13-actin using the 2-ΔΔCt method. Resultsrepresent mean±standard error and statistical tests were conducted usinga one-way ANOVA followed by Tukey's multiple comparisons test.

To test the possibility that Tonabersat-mediated reduction in Cx43plaques seen in FIGS. 10 and 12 was caused by down-regulation in Cx43mRNA transcription, we used real time reverse transcriptase-PCR tocompare the relative differences Cx43 mRNA following 1 hour treatmentwith tonabersat. There was no significant difference in Cx43 mRNA inARPE-19 cells following treatment with 50 μM tonabersat compared to bothuntreated (p=0.7572) and vehicle controls (p=0.10245) (FIG. 13).

Example 8: Tonabersat does not Affect hCMVEC Cell Viability MTT Assayfor Cell Viability

Stock MTT was prepared by dissolving 5 mg in 1 mL PBS producing aconcentration of 5 mg/mL. After the treatment incubation period of cellsseeded in a 96 well-plate (at 1×104 cells/well) the media was removedand replaced with 100 μL of PBS. 10 μL of MTT stock solution was addedto each well, except for those that were used as a blank, and left toincubate at 37° C. for 4 hours at which time the solutions was aspiratedfrom each well. Then 50 μL DMSO (Sigma) was added to each well todissolve the crystalline product. Viability was determined by measuringthe absorbance of the purple crystal produced. This was measured byreading the absorbance at 595 nm in a plate reader; the data wasstandardized to each control as an internal standard. Statistically wasinvestigated using One-way ANOVA and Tukey's post hoc test where*p<0.05.

The effect of tonabersat (0.2 μM to 200 μM) on hCMVEC cell viabilityover 24 hours was examined. Tonabersat (200 μM) did not significantlyreduce cell viability after 24-hour treatment (p=0.378), indicating alack of toxicity. Furthermore, there was no significant reduction incell viability in response to vehicle (0.2% DMSO) or carbenoxolone (200μM) (p=0.9029, p=0.4719) (FIG. 14B).

Example 9: Tonabersat Prevents Retinal Ganglion Cell Loss in RetinalIschemia-Reperfusion In Vivo Retinal Ischaemia-Reperfusion Model andTreatment

Animals were anesthetized with an intraperitoneal injection of ketamine(60 mg/kg) and medetomidine hydrochloride (0.4 mg/kg) and the corneaanaesthetized with oxybuprocaine hydrochloride (0.4%). The technique ofretinal ischaemia-reperfusion has previously been described (D. Sun, etal., The Journal of comparative neurology 505, 114 (Nov. 1, 2007)).Briefly, the left anterior chamber was cannulated with a 30-gaugeinfusion needle connected by silicone tubing to a reservoir of sterile0.9% saline. Cannulation was performed using a stereotaxic manipulatorarm to avoid injury to the corneal endothelium, iris, or lens. Theintraocular pressure of the cannulated eye was raised to 120 mmHg for 60minutes by elevating the saline reservoir. Retinal ischaemia-reperfusionwas confirmed by pallor of the posterior segment. After 60 minutes, thecannula was removed and reperfusion of the retinal vessels was confirmedby ophthalmoscopy. Three experimental arms were applied at the end of 60minutes of ischaemia. These included: no treatment (n=6), 10 mg/kgTonabersat (n=6), or 1 mg/kg Tonabersat (n=4). Systemic delivery of 0.25mL of a 10 mg/ml or 1 mg/ml Tonabersat solution diluted in 40% PEG, 60%cyclodextrin solution was achieved through intraperitoneal delivery atthe start of reperfusion. A final blood Tonabersat concentration of0.319 mM or 0.0319 mM was intended, assuming a blood volume of 20 mL andtotal systemic uptake of peptide. Animals were euthanized with CO₂ at 7days after reperfusion. One uninjured animal was euthanized to obtainuninjured retinas as normal controls.

After euthanasia eyes were enucleated, and the cornea, lens, andvitreous humour removed. The dorsal aspect of the retina was notched toretain orientation. The retina and sclera were fixed in 4% PFA in PBSfor 60 minutes at room temperature (RT). The retina was then carefullyremoved from the sclera and permeabilized via incubation in 0.5%Triton-X100 in PBS solution for 15 minutes at −80° C. Following thoroughwashing with PBS, free floating retinas were incubated overnight at 4°C. in goat anti-Brn3a primary antibody (SC-31984, Santa-CruzBiotechnology, 1:100) in 2% horse serum and 2% Triton-X100 in PBSsolution. After further washing of the retinas with PBS a donkeyanti-goat Cy3 secondary antibody (705-165-147, Jackson Immuno Research,1:500) solution was applied for 2 hours at RT. Retinas were mounted ontoSuperFrost Plus slides using citifluor mounting medium, and imaged.

Imaging and Quantification

Two fields in each quadrant of each retina were imaged giving a total ofeight images per retina. This method insured similar locations wereassessed between different eyes and avoided any possible area biaspresent in the retina. Retinal ganglion cell (RGC) labelling was imagedat 10× magnification. Voltage and offset settings were adjusted to bestdiscriminate individual antibody labelling and to avoid oversaturationof the image. Quantification was performed using automated spot countsin NIH ImageJ software. For RGC quantification each image was firstfiltered with a sharpen filter to delineate cell edges before beingconverted to a binary image using a threshold of 33. Spots of one or twopixels that resulted from noise and artefacts that were clearly not RGCof origin were excluded during particle counts. RGC density wascalculated as the number of RGCs per cm2.

Prevention of Retinal Ganglion Cell Loss in Retinal Ischemia-Reperfusion

A low concentration of tonabersat is protective against retinal celldeath as shown by an established in vivo model of retinalischemia-reperfusion (H. V. Danesh-Meyer et al., Brain 135, 506(February, 2012). A statistically significant (p<0.01) 31% loss of RGCswas found in ischemic eyes with no treatment (167111±14188 cells/cm2,mean±standard error) compared to normal controls (242558±15840cells/cm2) (FIG. 15). A statistically significant (p=0.03) 35% RGC losswas found in ischemic eyes treated with 10 mg/kg Tonabersat(158739±26370 cells/cm2). However, ischemic eyes treated with 1 mg/kgTonabersat had reduced neuronal loss (18%, 199927±26058 cells/cm2)compared with the other groups. Although not reaching statisticaldifference for improvement over injured but untreated eyes, thedifference between this group and the normal controls was no longerstatistically significant (p=0.17). No loss of RGCs was found in allcontralateral eyes.

Example 10: Tonabersat can Uncouple Gap Junctions in a Concentration andTime-Dependent Manner

An in vitro scrape-loading assay with extracellular Lucifer Yellow (LY),a fluorescent dye that is transferred only through coupled GJs wasperformed to test whether Tonabersat inhibits GJ communication (M. H.el-Fouly, et al., Experimental cell research 168, 422 (February, 1987)).A single scrape wound was made to a confluent monolayer of hCMVEC cellsto enable LY dye to gain access to the cytoplasm of cells at the edge ofthe wounded region and allow the dye to transfer to adjacent cellsthrough GJs. As expected, we observed LY transfer in vehicle control(FIG. 16A), which is indicative of functional cell-to-cell communicationvia coupled GJs (el-Fouly, 1987). Carbenoxolone, a broad spectrumgap-junction blocker, significantly reduced GJ communication down to77.8±7.4% compared to control (p<0.0001) (FIG. 16B). In Tonabersat theextent of GJ uncoupling increased over time (FIG. 16B). LY positivecells were significantly down to 72.5±7.4% at 2 hours (p=0.0132), andwas reduced to 62.8±7.4% by 6 hours (p=0.0003) compared to control. LYpositive cells was maximally reduced to 50.5±8.4% (p<0.0001) compared tocontrol by 24 hours in 50 μM Tonabersat (FIG. 12B). At a higherconcentration of 100 μM Tonabersat, LY positive cells were furthersignificantly reduced to 46.7±7.8% (p<0.0001). However, 50 μM Tonabersatimmediately (p=0.986) or 1 hour prior to the assay (p=0.054) did notsignificantly reduce GJ coupling when compared to control (FIG. 16B). Atlower Tonabersat concentrations (0.1, 1 and 10 μM), there was nosignificant reduction in LY positive cells at both 2 hours (p>0.7), and24 hours (p>0.3) compared to control (FIG. 16C,D). Taken together, theseresults indicate that Tonabersat mediated uncoupling GJs isconcentration-dependent (≥50 μM), and the extent of this uncouplingeffect is dependent on the time period of Tonabersat treatment (at thehigher uncoupling doses).

Discussion

These examples show the discovery of novel mechanism of actions oftonabersat that involves of direct and immediate block of Cx43hemichannels, and a concentration- and time-dependent reduction in GJcoupling. Short-term exposure to certain, lower concentrations ofTonabersat (10 μM) effectively inhibited Cx43 hemichannel mediated ATPrelease during injury and reperfusion in vitro, and this was consistentwith reduced RGC loss in an established in vivo retinalischaemia-reperfusion model. However, exposure to higher concentrationsof Tonabersat (<100 μM) not only uncoupled GJs, but a more sustained,longer-term treatment can elicit the targeted internalization anddegradation of Cx43 plaques via the lysosomal pathway.

It has been surprisingly discovered that tonabersat exhibits aconcentration-dependent inhibition of connexin hemichannels. A lower, 10μM concentration was the more effective tonabersat concentration forhemichannel inhibition, which exceeded the efficacy of peptide5 used inthis study. Furthermore, the inhibition was selective tohemichannel-mediated ATP release as a combined probenecid treatment wasrequired to observe the inhibitory effects of tonabersat (FIG. 8B).These data suggest that while tonabersat effectively blockshemichannels, it and its analogues and other compounds of Formula I areunlikely to inhibit ATP released from pannexin channels in ischemicinjury. If tonabersat effectively inhibited both connexin hemichannelsand pannexin channels, the level of the inhibition would have beencomparable to the combined peptide5 and probenecid treatments or CBX(55% vs. 65%, respectively). Conversely, tonabersat treatment aloneinduced a slight increase in ATP compared to injury alone, albeit notsignificant. It is also of note that 100 μM Tonabersat was not aseffective in lowering ATP as 10 μM Tonabersat in these experiments. Ashigher tonabersat concentrations was not linked to cell toxicity (FIG.14), tonabersat may have dual effects during injury whereby hemichannelsare targeted for inhibition, but pannexin channels may be triggered toopen in an off-target effect. These data indicate that a modestdose/concentration of tonabersat combined with a pannexin channelblocker such as probenecid will be effective as a treatment forischemia, and diseases, conditions and disorders that are characterizedat least in part by ischemia.

It has also been demonstrated herein that post-ischemic (reperfusion)conditions in vitro of treated models return to normal physiologicalconditions post 2 hours in HAIR solution. The in vitro model enabled theinventors to test a) the contribution of connexin and pannexin channelsnot only in ischaemic injury but also post-ischaemia b) the effect ofTonabersat treatment in ischaemia and post-ischaemia. Duringpost-ischaemia, connexin hemichannel accounted for almost all ATPrelease as shown by 100 μM peptide5 and 1 mM probenecid (FIG. 9).Importantly tonabersat at 10 μM concentration showed efficacy that wascomparable to 100 μM peptide5. These data suggest that a lowerconcentration of tonabersat inhibits connexin hemichannel-mediated ATPrelease post-ischemia.

Furthermore, it has also surprisingly been discovered that both connexinhemichannel opening and pannexin channels contribute to ischemic injury(FIG. 8) by mediating ATP release, but following reperfusion pannexinchannels may close and connexin hemichannels alone appear to account foralmost all of the ATP release through these two channel types (FIG. 9).Pannexin channels may open in an ischemia-dependent mechanism (i.e.NMDAR), but may close shortly after reperfusion when these stimuli alsosubside. In contrast, Cx43 expression was shown to peak at 4 hours withpeptide5 reducing vascular damage 4 and 24 hours post retina ischaemia(H. V. Danesh-Meyer et al., Brain: a journal of neurology 135, 506(February, 2012).) This is supported by reports that peptide5 delivered1 hour before and during ischaemia in near-term fetal sheep had minimaltherapeutic benefit (J. O. Davidson, et al., Experimental neurology 248,301 (October, 2013)), whereas peptide5 post-ischemia significantlyimproved the recovery of EEG activity and sleep cycling, andsignificantly reduced seizures.

In a previous study, a model of retinal ischemia-reperfusion injury wasinvestigated wherein a breach in the blood-brain barrier and edema ofthe vascular endothelium indicated the inflammatory response and causeRGC death (H. V. Danesh-Meyer (2012)). The cause of the injury wasattributed to open Cx43 HCs, given a dose of Cx43 mimetic peptide5 thattargeted Cx43 HCs but not GJs (S. J. O'Carroll, et al., Cellcommunication & adhesion 15, 27 (May, 2008)) significantly reducedvascular leak and RGCs cell death.

The Examples herein include the in vivo retinal ischemia-reperfusioninjury model, as carried out in Danesh-Meyer et al. (2012), and showedthat tonabersat is protective against RGC loss following retinalischemia-reperfusion injury. This was achieved at a low finalcirculating dose of 32 μM, comparable to 10 μM used for inhibitingconnexin hemichannels mediated ATP release in vitro.

Tonabersat has been administered in most Phase 2 clinical trials at20-80 mg once daily for migraine (A. W. Hauge, et al., The Lancet.Neurology 8, 718 (August, 2009)) and epilepsy (A. A. Parsons et al.,British journal of pharmacology 132, 1549 (April, 2001), N. Upton etal., British journal of pharmacology 121, 1679 (August, 1997)). However,those clinical results are inconclusive (Hauge, 2009). In contrast, ithas been surprisingly demonstrated herein that that the therapeuticbenefits can be achieved by targeting hemichannels during and afterinjury, especially after ischemia or in combination with a pannexinchannel blocker. The clinical failure may be attributed to use oftonabersat where pannexin channels were also affected.

Cx43 isoform specific antisense oligodeoxynucleotide (Cx43 AsODN)transient down-regulation significantly provides vascular protection andimproves functional outcomes in non-healing ocular wounds (Ormonde etal., J. Membr. Biol. (2012) 245(7):381-88). In this context, transientlycounteracting the rise in Cx43 and hence de novo formation of GJhemichannels was beneficial without permanently compromising the spatialbuffering effect. It has been surprisingly discovered herein thattime-dependent action of tonabersat may be utilized for controlledtransient modulation of Cx following injury whilst minimizing thecomplete removal of the ‘spatial-buffering’ effect.

This present disclosure also demonstrates that tonabersat removes Cx43from the plasma membrane (FIG. 16). The inventors have surprisinglydiscovered a reduction in the total expression of Cx43 plaques (FIG.16), which indicates that tonabersat is likely to alter the balancebetween Cx43 synthesis and degradation. Therefore, internalized Cx43channels targeted by tonabersat are sent for degradation via thelysosomal pathway.

There are two major routes for degradation of connexin channels, thelysosomal and proteasomal pathways. The rationale for examining thelysosomal pathway is based on the idea that plasma membrane connexinsare solely degraded by the lysosomes whereas cytosolic and nuclearconnexins are degraded by proteasomes. The accumulation of cytoplasmicCx43 in the presence of lysosome inhibitor indicates that Tonabersattargets Cx43 for degradation. The perinuclear localization of Cx43 isindicative of accumulation of new protein in the Golgi, which furthersupports the idea that while tonabersat enhances the degradation of cellsurface Cx43 channels, it has no effect on Cx43 transcription ortranslation of new protein. The PCR data also suggest that tonabersatdoes not regulate Cx43 mRNA transcription; rather, the observedreduction in Cx43 occurs at the level of protein turnover byinternalization and degradation.

In conclusion, the inventors have surprisingly discovered, amongst otherthings, novel mechanism of actions of tonabersat that include direct andimmediate block of connexin hemichannels, and, at higher concentrations,a concentration- and time-dependent inhibition of Cx43 GJ coupling.Exposure to high concentrations of tonabersat not only uncoupled GJs,but the sustained long-term treatment caused Cx43 plaques to be targetedfor internalization and degradation via the lysosomal pathway. In thepresent disclosure, it has been demonstrated that both connexinhemichannels and pannexin channels can mediate ATP release duringischemic injury, with connexin hemichannels being the primarycontributor to ATP leak post-injury. Short-term exposure to lowerconcentrations of tonabersat effectively inhibited Cx43 hemichannelmediated ATP release during injury but especially during reperfusion invitro, and this was consistent with reduced RGC loss in an in vivoretinal ischemia-reperfusion model, Tonabersat is thus not CNS specificas previously assumed, is not restricted to Cx26 modulation, and doesnot affect Cx43 protein synthesis, but rather acts directly on connexinhemichannels or through junction internalization.

Example 11: Transfection In Vitro Assay of Unmodified and ChemicallyModified Oligonucleotide Structures of Connexin 43 Antisense Compounds

HUVEC cells (C-003-5C, Invitrogen, primary Human endothelial cells) wereused to test the transfection of chemically modified backbones of theConnexin 43 antisense compounds. Cells were transfected withOligofectamine® using the manufacturer's recommended protocol for 4 hrand 12 hr in serum-free media. The antisense concentration was 200 nM inbuffer. The antisense compounds used had unmodified (O) or fullymodified (PTO) backbones, where the modified backbone had allthiophosphoriate linkages between the nucleotides. The unmodifiedsequence SEQ1-O would have the sequence GTAATTGCGGCAAGAAGAATTGTTTCTGT(SEQ ID NO: 23). The modified sequence SEQ1-PTO would have the sequenceof: GsTsAsAsTsTsGsCsGsGsCsAsAsGsAsAsGsAsAsTsTsGsTsTsTsCsTsGsT (SEQ IDNO: 23), wherein the subscript “s” indicates the thiophosphoriatelinkage between the two nucleotides. Likewise, the unmodified andmodified sequences for SEQ4-O and SEQ4-2PTO would beACCCATGTTGCCTGGGCACC (SEQ ID NO: 5), andAsCsCsCsAsTsGsTsTsGsCsCsTsGsGsGsCsAsCsC (SEQ ID NO: 5), respectively.All sequences for the transfection study were labelled with FAM forfluorescence labelling. The transfected cells were analyzed by FACS(fluorescent-activated cell sorting), and confocal microscopy.

Results:

Over 95% transfection efficiency was observed with all oligonucleotidetypes. Cells accumulate more PTO-oligonucleotides compared tononmodified oligonucleotides. Most uptake of the antisenseoligonucleotides takes place within 4 hours, as summarized in Table 40.

TABLE 66 4 hr post-transfection % Transfected SEQ4-O SEQ4-PTO SEQ1-OSEQ1-PTO Total 99 100 99 100 M2 64 9 56 26 M3 34 44 42 63 M4 1 48 2 11

TABLE 67 12 hr post-transfection % transfected SEQ4-O SEQ4-PTO SEQ1-OSEQ1-PTO Total 99 99 98 100 M2 55 9 56 15 M3 41 44 40 49 M4 2 47 2 35

FIG. 17 shows the FACS data for the transfection uptake of severalselected antisense oligonucleotides.

The image of live cells via confocal microscopy shows uptake ofFAM-labelled SEQ1-O (unmodified) in HUVEC cells (4 hrpost-transfection), as shown in FIGS. 18 and 19. FIG. 18 shows the imageof live cells via confocal microscopy shows uptake of FAM-labelledSEQ4-PTO (modified) in HUVEC cells (4 hr post-transfection). Greenrepresents the oligo SEQ1-0 (FAM labelled), Blue represents cell nucleusstained by DAPI. As can be seen in FIG. 18, the labelled connexin 43modulator is in close proximity to the nucleus and occasionally in thenucleus as shown in FIG. 18.

The oligonucleotide (green) is observed to accumulate in the nucleus(blue), as shown in FIGS. 18 and 19. In FIG. 19, green represents theoligo SEQ1-0 (FAM labelled), and blue represents cell nucleus stained byDAPI.

Next, the knockdown efficiency of Connexin 43 in HUVEC cells wasmeasured by qPCR for an oligonucleotide series with the same sequencebut with a different number of PTO-modifications on the oligonucleotidebackbone. The sequences tested were SEQ1 and SEQ4, with none, 2, or allphosphorthioate linkages in the backbone. For the sequences with twophosphorthioate linkages in the backbone, the phosphorthioate linkageswere between the last nucleotide linkages at the terminus of each sideof the sequence. The sequences are GsTAATTGCGGCAAGAAGAATTGTTTCTGsT (SEQID NO: 23) and AsCCCATGTTGCCTGGGCACsC (SEQ ID NO: 5) for SEQ1-2PTO andSEQ4-2PTO, respectively. The concentration of all oligo solutions was200 nM.

FIG. 20 shows the knockdown efficiency of Connexin 43 as measured byqPCR (n=3). The data shows the standard deviation for 3 replicates. FIG.21 shows the knockdown efficiency of Connexin 43 as measured by WesternBlot. The data show standard deviation for 3 replicates.

The results show that the full PTO backbone exhibited the most Cxn43knockdown by both qPCR and Western blot analyses. Furthermore,protecting the antisense with one PTO link on both ends (2-PTO) resultedin some degree of Cxn43 knockdown. Unmodified (0) backboneoligonucleotides resulted in much lower levels of Cxn43 knockdownefficiency than all PTO-backbone oligonucleotides.

The knockdown efficiency of modified (PTO) and unmodified sequences,both the full intact sequence and for scrambled sequences, was observedover time. The knockdown efficiency was measured by qPCR, as shown inFIG. 22. The data show standard deviation for 3 replicates.

FIGS. 22 A and B shows the knockdown efficiency of modified (PTO) andunmodified sequences measured by qPCR at 4 hours and 8 hours aftertransfection, respectively. Oligo concentration: (1)=200 nM; (3/4)=150nM; (1/2)=100 nM; C: SEQ1; SEQ4: 37501; Cscr: SEQ1 scrambled; LP2scr:SEQ4 scrambled; Orange bars: negative controls. (n=3 for each result).The results show lesser non-specific negative control effects detectedat 4 hr post-transfection, and the oligo at 150 nM exhibited the largestdifference from its corresponding negative control.

FIG. 23 shows the knockdown efficiency as measured by Western blot. Thedata show standard deviation for 3 replicates. The results show 4 and 8hr post-transfection exhibited very little knockdown, lessernon-specific negative control effects were detected at 12 hrpost-transfection, and the oligo at 150 nM concentration exhibited thelargest difference from its corresponding negative control.

Different sequences were analyzed by qPCR for their knockdown efficiencyof connexin 43 to compare sense strands and other antisense strands toCxn43, as shown in FIG. 24.

The sequences were also analyzed by Western blot (WB) for knockdownefficiency, as shown in the FIG. 25. The results indicate that all theantisense oligos had higher knockdown efficiency of Connexin 43 than thenegative controls. From the knockdown efficiency results, a variety ofother sequences (all with all-thiophosphoriate linkages in the entirebackbone) were identified that could also knockdown Connexin 43. Table68 shows their relative knockdown difference compared to SEQ1.

TABLE 68 SEQ # Absolute Difference from SEQ1 SEQ4 22 SEQ11 20 SEQ5 19SEQ6 18 SEQ7 17 SEQ8 15 SEQ9 14 SEQ10 12

The oligonucleotide sequences were then tested for their knockdownefficiency in another cell type: PAOEC (P304-05, Cell ApplicationsInc.), Pig Aortic endothelial cells, as primary cells. The cells wereseeded for 24 hr to reach 50-80% confluency prior to experiment. Cellswere transfected by Oligofectamine® using the recommended protocol at200 nM oligo concentration for 4 hr in serum-free media. Samples wereharvested at 8 hr (for qPCR analysis) and 12 hr (for western blotanalysis). FIG. 26 shows the knockdown results.

The dashed line in FIG. 26 indicates the mean negative control effect(˜12%). The results show that in general, pig version antisense oligosperformed better than the corresponding human versions.

Selected oligos were then analyzed for their dose-response performancein HUVEC cells by qPCR. The concentrations tested were: 1, 5, 10, 25,50, 100, and 200 nM, as shown in FIG. 27. The results show that thescrambled sequence (not antisense for Connexin 43) did not have anydose-response, whereas the selected antisense oligo sequences (all withphosphorthioate backbones) did exhibit a dose-response.

Knockdown efficiency of Cxn43 by the unmodified sequences (nophosphorthioate linkages in the backbone) was measured by qPCR forseveral sequences, as shown in FIGS. 28 A and B. The results indicateknockdown is detected at 4 hr but not at 8 hr post-transfection. Theantisense Connexin 43 oligos exhibited a dose response for SEQ4 and133704. SEQ1 did not exhibit a knockdown effect over ˜10% at 4 hr forall concentrations. The negative controls exhibited higher Cxn43 mRNAlevels compared to vehicle control.

The knockdown efficiency of the modified (with all-thiophosphoriatebackbone) sequence was compared to the unmodified sequence at both 400nM and 150 nM, as shown in FIG. 29.

The results indicate that at 200 nM, not much knockdown (KD) is detectedwith ASN treatment, while at 400 nM, low levels of knockdown aredetected for some ASN sequences. Overall, the unmodified oligo sequencescan induce knockdown in vitro but the extent is small and variable fromexperiment to experiment, as shown in FIG. 30.

The results indicate the modified oligo backbone (PTO-ASN) exhibitedmuch more knockdown efficiency than the corresponding unmodified oligo(O-ASN), even though a much higher dose of the unmodified oligo wasused.

Example 12. Other Sequences Identified by a Series of Screening Eventsto Identify Other Effective Antisense Oligonucleotides to Connexin 43

The DNA sequence of the target-of-interest was obtained from the NCBIdatabase (National Center for Biotechnology Information). DNA wassynthesized and inserted into the pDONR221 cloning vector foramplification using E Coli cells. Subsequently, the vector containingthe target DNA was extracted from the E Coli cells and the target DNAwas isolated using restriction enzymes. The target DNA was used as thetemplate for in vitro transcription to generate the corresponding RNAand used for performing RNAse H and DNAzyme assays.

For RNAse H assay, target RNA was labeled with ³²P at one end and probedwith a 12-nucleotides randomized oligonucleotide library in the presenceof RNAse H enzyme. Subsequently the partially digested sample productwas analyzed by performing a denaturing polyacrylamide gelelectrophoresis. The size of the fragments generated by RNAse H cleavagecompared to a standard ladder indicated the distance of thecorresponding accessible regions from the labeled end of the RNA.Regions that were situated on the untranslated ends of the RNA were notdetermined due to the resolution limit of the gel.

For DNAzyme assays, 20-nucleotide DNAzymes were designed by looking forthe enzymatic targets of purine (A, G) and pyrimidine (C, T) pairs onthe target RNA, in order to probe the specific accessible sites.Subsequently the samples were analyzed by performing non-denaturingagarose gel electrophoresis and RNA gel stain. The size of the fragmentsgenerated by DNAzyme cleavage confirmed the specific accessible sites.Sites that were situated on the untranslated ends of the RNA were notdetermined due to the resolution limit of the gel.

The sequences of the target binding arms of active DNAzymes are used tosynthesize antisense oligonucleotides with phosphorothioate (PTO)backbones—which were then screened for knockdown of the protein byWestern blotting and of the RNA by qPCR in cell culture assays.

Cells were grown in 6-well culture plates for 24 hr and reached 50-70%confluence prior to antisense oligonucleotide treatments using thetransfection agent Oligofectamine®. Cells were harvested for qPCR andWestern blotting analysis between 4 and 24 hr after transfection.

qPCR was performed to determine the efficacy of antisenseoligonucleotide in knockdown of the target RNA. Briefly, total RNA wasextracted from the cells after transfection. The isolated RNA wasDNAse-treated and reverse-transcribed into cDNA. The cDNA subsequentlyunderwent qPCR thermal cycling. The Ct values (the number of cyclesrequired for the fluorescent intensity to cross the set threshold)obtained from the qPCR were converted into relative abundance of RNA.The RNA level of the target was normalised to selected reference genesand compared between antisense oligonucleotide versus vehicletreatments.

Western blotting was also performed to determine the efficacy ofantisense oligonucleotide in knockdown of the target protein. Briefly,total proteins were extracted from the cells after transfection and theconcentration was determined by performing bicinchoninic acid (BCA)assay. Subsequently, equal amounts of total protein were loaded for gelelectrophoresis and blotted onto nitrocellulose membranes, which werethen probed with a specific antibody against the target protein.Densitometry was used to quantify the band intensity of the targetprotein. Knockdown was quantified by comparing antisense oligonucleotideversus vehicle treatments. Table 56, set forth in the Appendix inApplication Ser. Nos. 62/080,217, 62/085,226. 62/146,128, and62/147,488, incorporated here by reference, showed all ASN tested, andthe summarized test results.

Table 67 shows the sites where the RNAase enzyme activity was found andrepresents general regions of accessibility. These were refined by aDNAzyme walk across those regions. From this a range of ASN weredesigned and tested.

Table 68 lists all the ASN that were then tested on cells for knockdownactivity.

Table 61 lists the current lead ASN sequences we are further developingand characterising.

Results

These ASN sequences resulted from DNAzymes designed around 8 accessibleregions which were identified by the RNAse H assay, as listed in Table57.

TABLE 57 SEQ IDENTI- ASN FIER- target ASN NOT A SEQ SEQ Sense site codeSEQ ID ID ASN ID sequence from name NO. NO: sequence NO: (target) 5′ end24501 SEQ 122 24 ACCCATG 48 GGTGCCC 237-256 TTGCCTG AGGCAAC GGCACCATGGGT 24502 SEQ 123 25 GTTGCCT 49 AAGAGTG 231-250 GGGCACC GTGCCCAACTCTT GGCAAC LP1 SEQ 13 26 GCCTGGG 50 CAAAAGA 228-247 CACCACT GTGGTGCCTTTTG CCAGGC 24503 SEQ 124 27 TGGGCAC 51 AGCAAAA 226-245 CACTCTTGAGTGGT TTGCTT GCCCAG 30004 SEQ 125 28 GTAGGCT 52 CTTGACA 281-300TGAACCT AGGTTCA TGTCAA AGCCTA 37503 SEQ 126 29 TCTCCCC 53 GTTGAGT372-389 AGGCTGA CAGCCTG CTCAAC GGGAGA SEQ4/ SEQ 127 30 CCAGGCT 54CAGCGGT 368-385 37501 GACTCAA TGAGTCA CCGCTG GCCTGG 37502 SEQ 128 31CTCAACC 55 TGCTGGG 357-374 GCTGTCC GACAGCG CCAGCA GTTGAG 47001 SEQ 8 32CAGAAGC 56 ATCTCTC 464-483 GCACATG ATGTGCG AGAGAT CTTCTG 47002 SEQ 11 33GAAGCGC 57 CAATCTC 462-481 ACATGAG TCATGTG AGATTG CGCTTC 50501 SEQ 12934 AGTGTGG 58 TTTGTGT 500-519 GTACAGAC CTGTACC ACAAA CACACT 50502SEQ 130 35 TGGGTAC 59 CATATTT 496-515 AGACACA GTGTCTG AATATG TACCCA50503 SEQ 131 36 GGTACAG 60 ATCATAT 494-513 ACACAAA TTGTGTC TATGATTGTACC LP3 SEQ 12 37 CAGACAC 61 GCAGATC 490-509 AAATATG ATATTTG ATCTGCTGTCTG 50504 SEQ 132 38 GACACAA 62 CTGCAGA 488-507 ATATGAT TCATATTCTGCAG TGTGTC 50505 SEQ 133 39 ACAAATA 63 GTCCTGC 485-504 TGATCTGAGATCAT CAGGAC ATTTGT 50506 SEQ 9 40 ATATGAT 64 CTGGGTC 481-500 CTGCAGGCTGCAGA ACCCAG TCATAT 87201 SEQ 134 41 ATGATGA 65 GAGAAAA 863-882AGATGGT CCATCTT TTTCTC CATCAT 87202 SEQ 135 42 ATGAAGA 66 ACCATCT869-888 TGATGAA TCATCAT GATGGT CTTCAT 87203 SEQ 136 43 AGCATGA 67ATCTTCA 872-891 AGATGAT TCATCTT GAAGAT CATGCT 87204 SEQ 137 44 ATGAAGA68 ACGGAGA 860-879 TGGTTTT AAACCAT CTCCGT CTTCAT 1233 SEQ 7 45 AGGCTGT69 CTAACTC 1233- GCATGGG CCATGCA 1252 AGTTAG CAGCCT 133704 SEQ 6 46CGCTGGT 70 CTAGCCA 1316- CCACAAT TTGTGGA 1335 GGCTAG CCAGCG 133705 SEQ 547 GCTGGCT 71 GACCTTC 1335- CTGCTTG AAGCAGA 1354 AAGGTC GCCAGC

A subset of ASN was then chosen to take forward for further analysis.Other ASN were excluded from this based on showing lower levels ofknockdown in preliminary cell screening assays, and/or if highcross-reactivity was detected by sequence alignments with other knownhuman genes.

TABLE 58 List of ASN subset used in furtheranalysis due to promising profile ASN target ASN site codeSEQ ID NO. for ASN from name ASN sequence sequence 5′ end 24501SEQ ID NO.: 5 ACCCATGTTG 237-256 CCTGGGCACC 30004 SEQ ID NO.: 6GTAGGCTTGA 281-300 ACCTTGTCAA 37503 SEQ ID NO.: 7 TCTCCCCAGG 372-389CTGACTCAAC LP2/ SEQ ID NO.: 4 CCAGGCTGAC 368-385 37501 TCAACCGCTG 47001SEQ ID NO.: 8 CAGAAGCGCA 464-483 CATGAGAGAT 47002 SEQ ID NO.: 9GAAGCGCACA 462-481 TGAGAGATTG 50501 SEQ ID NO.: 10 AGTGTGGGTA 500-519CAGACACAAA LP3 SEQ ID NO.: 11 CAGACACAAA 490-509 TATGATCTGC 50506SEQ ID NO.: 12 ATATGATCTG 481-500 CAGGACCCAG 112304 SEQ ID NO.: 13GTAATTGCGG 1134-1153 CAAGAAGAAT 1233 SEQ ID NO.: 14 AGGCTGTGCA 1233-1252TGGGAGTTAG 133704 SEQ ID NO.: 15 CGCTGGTCCA 1316-1335 CAATGGCTAG 133705SEQ ID NO.: 16 GCTGGCTCTG 1335-1354 CTTGAAGGTC

The relative RNA knockdown activities of the antisense oligonucleotides(ASN) are shown in FIG. 31. The relative protein knockdown activitiesobtained from Western Blot analysis of the antisense oligonucleotidesare shown in FIG. 32. The results are from two replicate experiments inHUVEC cells. (n=2) The yellow bar (“47001scr2”) indicates the activityof the negative control oligonucleotide. The results indicate that otherASNs are highly efficient at regulating Cxn43 activity.

The cell screening experiments were performed using HUVEC cells (humanumbilical vein endothelial cells; Invitrogen), with two biologicalreplicates in each experiment. The identifiers of “CODA” are SEQ1, and“LP2” are SEQ4.

The following top ASN candidates were selected from those that gave apercentage knockdown within the top 10% detected in both the qPCR andWestern blot analyses, as shown in Table 61.

TABLE 61 Current top ASN candidates for Cxn43 Knockdown in HUVEC Avg %KD Sample qPCR WB SEQ4/37501 87 82  SEQ 5/133705 84 75  SEQ6/133705 8375 SEQ7/1233  82 75 SEQ8/47001 81 75 SEQ9/50506 79 73 SEQ10/37503  77 75

FIG. 33 shows a dose response curve of some selected ASNs to induce theknockdown of Cxn43 RNA at 4 hrs after administration to cells. Thescreening results are from one experiment in HUVEC cells. (n=2biological replicates). The grey line (“47001scr2”) indicates thenegative control oligonucleotide. The ASN concentrations were: 1, 5, 10,25, 50, 100, and 200 nM. The term “% KD” is the percentage of knockdownin Cxn43 RNA levels. The indicator “Coda” is CoDa001 human connexin 43ASN in clinical development; the indicator “scr2” is a scrambled ASNcontrol which is not expected to have Cxn43 Knockdown activity.

Example 13. Use of Connexin 43 Modulating Peptide to Prevent VascularLeak after Ischemia Reperfusion Injury

A connexin 43 (Cxn43) modulating peptide was used to control theincrease in ocular pressure of models induced with ischemia perfusion.The peptide sequence used was SEQ-pept5 (SEQ ID NO: 168), at a dosagedescribed below. Mice were subjected to ischemica perfusion, thentreated with Evans Blue Dye via intraperitineal injection to monitor thebaseline of the model. 100 mg/ml of Evans Blue in 0.9% saline solutionwas introduced into the mice, with 1 ml/100 g body weight dosage. Theinjections were performed intraperitoneally. The amount of dye leak wasmeasured, as was the connexin 43 spot count, followingischemia-reperfusion. FIG. 34 shows the dye perfusion of Evans Blue Dyepost-ischemia to map the connexin 43 following ischemia-reperfusion.

FIG. 35 shows the calculated (via ImageJ, as described herein) area ofdye leak or Connexin 43 spot count as a function of time followingischemia-reperfusion. The results show the baseline performance of dyeleakage.

The treatment of the ischemia-reperfusion model was analyzed todetermine the effect of treatment with an connexin 43 modulator. FIG. 36shows the effects on the total dye leak following treatment with, andwithout, the Cxn43 modulating agent. The Cxn43 modulating agent used inthis study was SEQ ID NO: 168 (“Peptide 5”). The injection wasintraperitoneal injection. 1 mL of a 2 mM Cxn43 modulator peptide wasused as a 0.9% saline solution. The final blood peptide concentrationwas 100 uM.

The peptide fragment efficiency was also monitored for a range ofsequence variations on a common peptide sequence core. As shown in FIG.37, the ability to retard the expression of Cxn43 (Y-axis) was comparedfor variants of a particular core sequence. The results indicated thatthe sequence must retain a KT at the carboxy terminus to retain activityin retarding Cxn43 activity. The peptides were administered at aconcentration of 100 uM.

The sequences in FIG. 37 are: Peptide 5=SEQ ID NO: 168, Mod 1=SEQ ID NO:171, Mod 2=SEQ ID NO: 172, Mod 3=SEQ ID NO: 173, Mod 4=SEQ ID NO: 174,Mod 5=SEQ ID NO: 175, Mod 6=SEQ ID NO: 176.

Example 14: Nanoparticle (Nps) and Microparticle (MPs) Preparation andAnalysis Particle Preparation

Water-oil-water (double) emulsion is one method by which PLGA can beused to encapsulate hydrophobic and hydrophilic drugs in micro- ornano-scale form. Briefly, PLGA (Poly(D,L-lactic-co-glycolic acid) (PLGA50:50, MW 13,600, inherent viscosity 0.19 dl/g, Sigma-Aldrich) wasdissolved into an organic phase (oil) that is emulsified with asurfactant or stabilizer (water). The hydrophobic forms of the connexin43 modulator were added directly to the oil phase, whereas hydrophilicforms of the connexin 43 modulator may be first emulsified with thepolymer solution prior to formation of particles. High intensitysonication bursts facilitated the formation of small polymer droplets.The resulting emulsion was then added to a larger aqueous phase andstirred for several hours, which allowed the solvent to evaporate.Hardened nanoparticles were collected and washed by centrifugation.

Preparation and characterisation of PLGA Nps and Mps PLGA Nps and Mpscontaining Cxn43 modulators were performed using the double emulsionsolvent evaporation method (Y.-S. Chen, et al., Drug Deliv. 18 (2011)493-50). Briefly, for Nps, 5 mM of Cxn43 MP (plain or FITC-labelled)aqueous solution was emulsified in DCM (dichloromethane, Sigma-Aldrich)containing 30 mg/ml of PLGA using a probe sonicator (Hielscher, Teltow,Germany) at an amplitude of 50 W and a duty cycle of 0.6 s for 1 min onan ice bath to form the primary w/o emulsion. The resulting primary w/oemulsion was further emulsified in an aqueous solution of 3% w/v PVA(polyvinyl alcohol (PVA, MW 130,000, hydrolysis degree 95-97%,Sigma-Aldrich) to form a multiple w/o/w emulsion. This was subsequentlypoured into 50 ml of an aqueous PVA solution (0.1% w/v) to stabilise thedouble emulsion during the evaporation process with the resultant Npsrecovered by ultracentrifugation (ProteomeLab™ XL-A/XL-I; Type 70 Tirotor, Beckman Coulter, Auckland, New Zealand) at 30,000 rpm at 4° C.For Mps, 10 mM of Cxn43 MP aqueous solution was emulsified in DCMcontaining 100 mg/ml of PLGA. The formed primary w/o emulsion wasfurther emulsified in a PVA solution (5% w/v) and then homogenised at8000 rpm for 5 min (IKA works, Ultra-Turrax T10, Wilmington, USA) beforepouring it into 50 ml of a 0.1% w/v PVA solution and evaporating theorganic phase at room temperature.

Nps and Mps were then lyophilised (VirTis, SP Scientific, Gardiner,N.Y., USA) for 24 h and the particle morphology was investigated usingscanning electron microscopy (SEM) (Philips XL30S FEG, Eindhoven,Netherlands), while the particle size, zeta potential (ZP) andpolydispersity index (PDI) were determined using a Zetasizer Nano andMastersizer (Malvern Instruments, Worcestershire, UK), respectively.

Entrapment Efficiency and In Vitro Cxn43 MP Release from PLGA Nps andMps

A 2.5 mg mass of FITC-labelled Cxn43 MP-loaded Nps and Mps was added to0.25 ml of DMSO to allow for polymer dissolution. Subsequently, 0.5 mlof water was added and the mixture was allowed to stand for 15 min atroom temperature before shaking for 30 min to facilitate extraction ofFITC-labelled Cxn43 MP into the aqueous phase. Samples were centrifugedat 13,000 rpm and 4° C. for 5 min and Cxn43 MP concentrations in thesupernatant were quantified using a microplate reader (Spectra Max M2Microplate Reader, Molecular Devices, Silicon Valley, Calif., USA). Forin vitro release studies, 15 mg of FITC-labelled Cxn43 MP-loaded Nps andMps was suspended in 0.7 ml of PBS and shaken at 100 rpm and 37° C. overthe period of the release experiment. A volume of 0.6 ml of supernatantwas withdrawn at pre-determined time points and replaced with freshmedium to maintain sink conditions. Samples were centrifuged at 13,000rpm and the supernatant was assayed for FITC-labelled Cxn43 MP using afluorometer as described above. Cumulative amounts of Cxn43 MP releasedwere calculated, with each study performed in triplicate.

In Vitro Block of Cxn43 Hemichannel Docking

ARPE-19 cells (P9-12, American Type Culture Collection (ATCC, Manassas,Va., USA) were initially assessed for the expression of Cxn43 protein.Cells were routinely cultured in DMEM/F12 (Gibco, NZ) containing 10% FCSat 37° C. with 5% CO2 and 95% relative humidity. Upon confluence, cellswere harvested with TrypLE™ Express and seeded onto coverslips at adensity of 1×105 cells/ml per well. After 48 h of adhesion, each wellwas washed with PBS three times, followed by addition of 1 ml ofFITC-Cxn43 MP solution or FITC-Cxn43 MP loaded Mps and Nps suspensions,which were freshly prepared by mixing the freeze-dried particles withcell growth medium, with the final Cxn43 MP concentration kept at 5micromolar (according to the previously confirmed entrapmentefficiencies). At predetermined time points (8 and 24 h), suspensionswere removed and cells were carefully washed with PBS three times toremove any residual particles. Cells were fixed with 4% cold PFA at roomtemperature for 20 min before incubation with a rabbit anti-Cxn43antibody (C6219, Sigma Aldrich, 1:2000) at 4° C. overnight. Afterremoving the primary antibody, cells were incubated with a goatanti-rabbit secondary antibody labelled with Alexa 568 (A11011,Invitrogen, 1:500) for 2 h. Nuclei were counterstained with DAPI(4′,6-diamidino-2-phenylindole, 100 nM in PBS) before mounting cellsonto slides using Citifluor mounting medium (AF1, ProSciTech, Sydney,Australia).

In Vivo Retinal Ischaemia—Reperfusion Rat Model

All procedures were compliant with the ARVO Statement of the Use ofAnimals in Ophthalmic and Vision Research and were approved by theUniversity of Auckland Animal Ethics Committee. Adult male Wistar ratsweighing 200-300 g were housed under a 12-h light/dark cycle andreceived food and water ad libitum. Animals were grouped according tohousing time periods after reperfusion and formulations applied (Table109).

TABLE 109 Number of animals (one eye per animal for each time period ofhousing post-ischemia and intravitreal injection Study Arm 28 d 90 dControl (no ischaemia) — — 1 h Ischaemia + Saline 3 (GFAP & — injection(vehicle control) Cxn43) + 6 (Brn3a) 1 h Ischaemia + Cxn43 MP 3 (GFAP &— solution Cxn43) + 6 (Brn3a) 1 h Ischaemia + Cxn43 MP 3 (GFAP & —loaded PLGA Nps Cxn43) + 6 (Brn3a) 1 h Ischaemia + Cxn43 MP 3 (GFAP & 3(GFAP & loaded PLGA Mps Cxn43) + Cxn43) + 6 (Brn3a) 6 (Brn3a) Cxn43MP,connexin43 mimetic peptide; Nps, nanoparticles; Mps, microparticles;GFAP, glial fibrillary acidic protein; Brn3a, brain-specifichomeobox/POU domain protein 3A.

Rats were anaesthetized with an intramuscular injection (0.1 ml/100 gbody weight) of Ketamine (100 mg/ml; Parnell Laboratories, Auckland, NewZealand) and Domitor (metadomidine hydrochloride 1 mg/ml; NovartisAnimal Health, Sydney, Australia) with a ratio of 6:4. A topicalanesthetic (Minims oxybuprocaine hydrochloride 0.4% w/v; Bausch & Lomb,Surrey, UK) was applied to the left eye to remove the corneal reflexprior to cannulation, while Refresh Tear Plus eye drops (Allergan,Auckland, New Zealand) were administered to the right eye (control) toprevent it from drying. Retinal ischaemia was achieved by cannulatingthe anterior chamber with a 30G needle inserted into the anteriorchamber of the left eye approximately 1 mm anterior to the limbus. Theneedle connected to silicone tubing was attached to an elevated infusionline of sterile 0.9% saline, with the height of the saline bagcalibrated to produce 120 mmHg. This state was maintained for 1 hfollowed by removal of the cannula, causing normalisation of the TOP andreperfusion of the retina. If any ocular complications such as lensdamage or excessive haemorrhage occurred as a result of the procedurethe animal was excluded from the study.

Intravitreal Injection of Cxn43 MP Formulations

Intravitreal injection was performed immediately followingischaemia-reperfusion. A Hamilton syringe (Model 701 LT SYR, HamiltonCompany, Reno, Nev., USA) with a 30G needle was inserted posterior tothe limbus in the superior retina at a 45° angle to avoid contact withthe lens capsule and to direct the contents into the vitreous chamber.Each rat received 2 μl of 580 micromolar Cxn43 MP diluted in 0.9%saline. A final peptide concentration of 20 micromolar was intended,assuming a vitreous volume of 50 μl. A volume of 0.1 ml/100 g bodyweight of Antisedan (atipamezole 5 mg/mL, Pfizer, Auckland, New Zealand)in saline (1:9) was then injected subcutaneously to allow the animal torecover.

Cxn43 and GFAP Labelling in Retinal Cross-Sections

After 28 d of reperfusion, rats were euthanized with CO2 and eyes wereenucleated (n=3 for each group). Eyes were immediately fixed bysubmersion in 4% PFA for 30 min, followed by overnight cryoprotectionwith gradually increasing sucrose from 10% to 30%. Eyes were thenembedded in optimal cutting temperature (OCT) compound (ProSciTech,Sydney, Australia) and snap frozen with liquid nitrogen. Sections of 20micrometer thickness were mounted onto slides and were further fixed in20° C. ethanol for 10 min before incubation with blocking buffer (10%normal goat serum (Gibco, NZ) and 0.1% Triton X-100 in PBS) for 1 h atroom temperature. A rabbit anti-Cxn43 (C6219, Sigma Aldrich, 1:2000) andmouse anti-GFAP conjugated to Cy3 (C9205, Sigma Aldrich, 1:1000) primaryantibody in 10% normal goat serum and 2% Triton X-100 were appliedovernight at 4° C. Tissues were washed with PBS three times for 15 min.A goat antirabbit Alexa488 secondary antibody (A11034, Invitrogen,1:1000) in 10% normal goat serum to visualise Cxn43 was applied for 2 hin the dark at room temperature before mounting the sections.

RGC Labelling in Retinal Wholemounts

After 28 and 90 d of reperfusion, rats were euthanized with CO2 and eyeswere enucleated (n=6 per group). The posterior segment of the eye wascarefully removed after cutting along the cornealscleral limbus junctionand the retina, sclera and optic nerve were fixed in 4% PFA in 0.01 MPBS for 1 h. A small cut was inserted to mark the superior quadrant andfour radial cuts were made around the circumference to divide thesuperior, inferior, temporal, and nasal retinal quadrants and allow flatmounting. The optic nerve and sclera were then gently detached and thefree retina was flattened onto slides carefully. Surviving RGC werelabelled by applying a goat anti-Brn3a primary antibody (SC-31984,Santa-Cruz Biotechnology, 1:100) in 2% horse serum (Gibco, Auckland, NewZealand) and 2% Triton X-100 in PBS at 4° C. overnight. Tissues werewashed with PBS three times for 15 min, before a donkey anti-goatCy3-tagged secondary antibody (705-165-147, Jackson Immuno Research,1:500) in 2% horse serum was applied for 2 h at room temperature in thedark. After further washing with PBS, retinas were flat-mounted andstored at 4° C. until imaged.

Confocal Microscopy and Image Analysis

Immunolabeling was imaged with a confocal laser scanning microscope(FV1000, Olympus, Tokyo, Japan). For Cxn43 and GFAP labelling in retinalcross-sections eight images were taken per sample with six sections usedper eye. For RGC labelling in retinal whole mounts, two fields perquadrant of each retina were imaged with six retinas used per group.This method insured that similar regions were assessed in each eye andavoided any possible area prejudice. Gain (voltage) and offset settingswere adjusted to best discriminate individual labelling and to avoidoversaturation of the image. Quantification was performed usingautomated spot counts in ImageJ. For Cxn43 quantification incross-sections, each image was converted to a binary image using athreshold of 45. Spots fewer than three pixels were considered as noiseand not counted. For RGC quantification in wholemounts each image wasconverted to a binary image using a threshold of 23. To separateclusters the watershed algorithm was applied. Spots of fewer than 20pixels were considered noise and were excluded from particle counts.

Statistical Analysis

Results were expressed as mean values with a standard deviation (SD).Analysis of variance (one-way ANOVA) with Tukey-Kramer comparison wasused to test for significant differences (p<0.05) between groups. Alldata analysis was carried out using Minitab# Statistical SoftwareVersion 15 (Minitab Inc., State College, Pa., USA).

Characterisation of PLGA Nps and Mps

Using the double emulsion and solvent evaporation method, Cxn43 MPloaded PLGA Nps and Mps of desirable sizes with narrow size distributionwere obtained (FIG. 110). FIG. 110 shows the morphology of thefreeze-dried Nps (A) and Mps (B) immediately after preparation as wellas the change in surface morphology and porosity after three days inrelease media (D (Nps) and C (Mps), respectively). As demonstrated, bothNps and Mps exhibited spherical structures and a relatively smooth andeven surface morphology after preparation (FIGS. 110A and B). Althoughthe formation of some agglomerates was observed, no free drug crystalswere present, indicating complete loading of Cxn43 MP into the PLGAmatrix. After three days in release media, Mps exhibited slight poreformation on the particle surface (FIG. 1C) while Nps were almostcompletely eroded with mainly particle capsules remaining (FIG. 1D).

The particle size was mainly influenced by the PLGA concentration. Thehigher the PLGA concentration used, the higher the mean diameterobtained as seen by the larger sized Mps. This is most likely due to thegreater probability of smaller polymer aggregates coalescing in a moreconcentrated solution, hence leading to larger droplets. Moreover, thehigher shear forces necessary during the emulsification process due tothe increasing viscosity of the organic phase may have resulted inpoorer dispersability of the PLGA solution into the aqueous phase.

The mean ZP (zeta potential) of Nps was −31.3±0.94 mV and since Cxn43 MPis neutral at pH 7.4 the negative charge was attributed to PLGA. The ZPis an important physicochemical characteristic as it influences bothparticle stability and mucoadhesion. Highly positive or negative ZPvalues cause repulsion between particles and therefore preventaggregation and thus in theory tend to stabilise particle suspensions.The ocular vitreous is composed of more than 98% water, with collagenand hyaluronan molecules comprising the two main solid components.Anionic particles may diffuse freely through the vitreous viaelectrostatic repulsion between the particles and the negatively chargedvitreous meshwork if their sizes are small enough to avoid stericaltrapping. The mesh pore size of bovine vitreous has been shown to bebetween 1 and 2 μm (L. Cheng, et al., J. Neurosci. 22 (2002) 3977-3986).Therefore, Nps will freely diffuse through the vitreous towards theretina, while Mps may get trapped in the meshwork due to their largersize.

Entrapment Efficiency and In Vitro Cxn43 MP Release from PLGA Nps andMps

The entrapment efficiencies (EE) of FITC-labelled Cxn43 MP loaded PLGANps and Mps are listed in FIG. 110. The reproducibility of the methodwas good, as indicated by the loading amount and entrapment efficiencycalculations were carried out on three batches per group prepared underidentical conditions. High yields (≥90%) were obtained for bothparticles, with Cxn43 MP EE being higher for the larger Mps (≥97%)compared to the much smaller Nps (≥70%), with an almost complete peptideencapsulation obtained for Mps. The relatively high concentration ofPLGA used for Mps contributed to the high EE by lowering the diffusivityof the active into the external water phase during processing.

Particle Sizing Analysis

Samples for Scanning Electron Microscopy (SEM) were prepared the day ofimaging. A strip of double-sided carbon tape was affixed to a SEM stub.A small quantity of lyophilized particles were applied to the surface ofthe tape, and the tape surface was gently subject to flowing nitrogen toremove loose particles. The sample was then sputter coated with gold orgold-paladium for 30-120 seconds. The SEM stub was then placed in theSEM for analysis. Typical parameters for visualizing particles were aworking distance of 5-15 mm, beam strength of 5-12 kV, and a spot sizeof 1-3 microns. Microparticles were observed at 100× magnification andnanoparticles were distinguishable at 3,000× magnification. At leastthree images were collected per batch in order to obtain arepresentative sample of particle size and morphology. Measurements weretaken directly from the SEM images using the “measure” function inImageJ. A minimum of 150 measurements of the diameter of particlesselected randomly from the field of view were taken to acquire arepresentative size distribution. Representative images of themicroparticles and nanoparticles are shown in FIG. 38.

Other methods may be employed to measure the particle size, includingbut not limited to light scattering, zeta potential analysis, coultercounting (electrical sensing zone method), and optical microscopy.

Example 15. Use of Microparticles and Nanoparticles for Extended ReleaseFormulations of Connexin Modulators

Microparticles and nanoparticles were loaded with the chemicallymodified C12-C12-SEQ-Pept5 connexin 43 modulator peptide (SEQ ID NO:237)via a method similar to that described above. To obtain a representativeimage of the particles after release media, an aliquot of each particletype was added to 1×PBS buffer for three days. The particles werecollected via a method similar to that described above, and imagedpost-release. The images are shown in FIG. 110 above. The particle sizeanalysis (as determined by the method described above, using ImageJparticle sizing of multiple SEM images) are given in the bottom of FIG.38 for the as-made particles (which were not yet subject to the releasemedia).

In vitro release of the connexin 43 modulator from the particles wasmonitored by UV-Vis of the solution over the particles. Particles wereadded to the release media, 1×PBS buffer, and aliquots of the buffersolution were monitored by UV-Vis to determine the amount of peptidereleased. The results are shown in FIG. 39.

The in vitro release results indicate that the particulate formulationsexhibit a sustained release of the connexin 43 modulator over time. Theinitial burst of modulator released is likely due to the modulatorphysiadsorbed to the particle surface, whereas the sustained release ofthe modulator is likely due to the diffusion of the modulator from thecore of the particles. The particle size is hereby an importantparameter, with the Mps exhibiting a relatively small initial burstcompared to Nps due to the decrease in surface area to volume ratio withincreased particle size. This resulted in reduced surface area availablefor the medium to penetrate into the particles and erode the polymer andtherefore slowed down the release rate of Cxn43 MP.

Example 16. Use of Chemically Modified Peptides and ParticulateSustained Release Formulations to Treat Glaucoma-Modelled Oculae

The effects of chemical modification and particulate formulationdelivery were measured on rats modelled for glaucoma with a retinalischemia reperfusion. First, a retinal ischemia reperfusion model wascreated. A total of 121 adult male Wistar rats weighing 200 to 300 gwere used for the study. Retinal ischemia was modeled in the left eye(120 mmHg for 60 min), with the right eye as the control, and then theconnexin 43 modulator peptide formulations were injected intravitreallyimmediately as solutions in 1×PBS delivery agent. The connexin 43modulator peptide used was SEQ-Pept5, in both its unmodified andchemically modified (didodecyl) forms. The formulations used were neat(just diluted in 1×PBS delivery media) solutions of nanoparticles ormicroparticles. After reperfusion, Evan's blue dye was injectedintraperitoneally 4 hr after reperfusion to visualize vessel leak usingconfocal microscopy. Retinal whole mounts were collected at 8 hr and 28days for Cxn43 quantification, and 28 and 90 days for gangliol cell(RGC) density quantification.

The results as shown in FIG. 40 indicate that the vessel leak after 4 hrpost-ischemia was significantly reduced in the retina for the eyestreated with any of the connexin 43 modulators tested. The results alsoindicated a reduction in vessel leak for the chemically modified(didodecyl) form of SEQ-Pept5 (C12-C12 Cxn43 MP) (SEQ ID NO: 237)compared to the unmodified peptide (Cxn43 MP) (SEQ-Pept5, SEQ ID NO:168).

Spot counts were measured for models treated with both forms of theconnexin 43 modulator peptide. As shown in FIG. 41, the resultsindicated that spot counts were reduced moreso with the chemicallymodified (C12-C12 Cxn43 MP) connexin 43 modulator peptide than theunmodified peptide. The lowest spot count was observed at just 8 hrafter ischemia.

After 28 days post-ischemia, cross-sections of the tissues were observedby confocal microscopy and the mean Cxn43 count was measured for Evan'sBlue dye-stained samples. The results, as shown in FIG. 42, indicatethat the formulations involving nanoparticles for the delivery resultedin lower Cxn43 expression than formulations without the nanoparticles,or even formulations with the microparticles. Also, over the extendedduration, both of the particulate formulations yielded a reduction inCxn43 expression compared to the initial ischemia timepoint.

Retinal ganglion cell count was also monitored for the models treatedwith the formulations involving nanoparticles, microparticles, or neat,and with the unmodified and chemically modified peptide. The results, asshown in FIG. 43, show that the chemically modified peptide treatmentresulted in over 93% RGC survival after 28 days compare to less than 70%for untreated eyes (ischemia only).

Example 17. Connexin 43 is Upregulated in the Retina of Humans Diagnosedwith AMD

Impairment of choridal perfusion and/or choroidal inflammation, orchoroidal overperfusion induces vascular leak in the choroid and Bruch'smembrane and results in endothelial cell loss in the retinal pigmentepithelium. Impairment of choridal perfusion and/or choroidalinflammation, or choroidal overperfusion and choriocapillaris dropoutresults from connexin 43 upregulation, and connexin 43 upregulation maybe a contributing cause of AMD. Abnormal vasculature was observed in thechoroid of AMD organ donor retinae associated with changes in Cxn43expression, supporting the role of connexin 43 upregulation in AMD. Thisindicates that Retinal Pigment Epithelium degeneration and Drusendevelopment are potentially down-stream consequences of AMD, rather thanprimary causes of AMD. Alterations in choroidal blood flow andimpairment of choridal perfusion and/or choroidal inflammation, orchoroidal overperfusion are merely contributory factors to AMD. Vascularleak and inflammation are associated with AMD choroidal changes whichare then subsequently associated with retinal neovascularisation.

As shown in FIG. 44, connexin 43 expression as observed in stainedsamples of human donors with AMD was upregulated in multiple areas ofthe choroid, retinal pigment epithelium, and associated areas adjacentto the Bruch's membrane. As observed in FIG. 44 Panel A, a non-AMD donorhad Cxn43 expression confined to the retinal pigmental epithelium. Therewas significant Cxn43 label in the retinal pigmental epithelium but thiswas masked by autofluorescence of the pigment itself. As observed inFIG. 44 Panel B, in the AMD diagnosed donor, extensive Cxn43 label wasobserved outside of blood vessels (within the extracellular matrix).This type of label is associated with extracellular matrix fibroblastsand inflammatory cell clusters, which was evident within areas of denserconnective tissue deposition.

The inventors have made the surprising development that regulation ofCxn43 in the retina is a potential treatment for AMD.

Example 18. Treatment of Acute Macular Degeneration (AMD) with Connexin43 Modulator Coadministered with an Anti-VEGF Compound

The excessive use of anti-VEGF reagents currently used to treat AMDleads to choriocapillaris dropout, thereby increasing incidence of thedisease. This was supported by the CATT 2 study showing increasedincidence of new geographic atrophy in patients treated long term withAvastin or Lucentis (Martin D F, Maguire M G, Fine S L, Ying G S, JaffeG J, Grunwald J E, et al. Ophthalmology; 119(7): 1388-98, 2012). Theincidence in patients treated monthly (average 23 injections over twoyears) with the smaller Lucentis molecule (where greater penetrationinto the choroid could be expected) was greater than in Avastin treatedpatients (29.4% versus 19.5% respectively). Patients treated as needed(average 12 or 24 injections over two years respectively) had lowerlevels of new geographic atrophy (16 and 14% respectively). Accordingly,penetration of AMD treatment into the choroid resulted in choroidinflammation, and damage to the vascular bed that nurtures the retina,cycling back into uncontrolled choroid inflammation. The inventors havemade the surprisingly discovery that breaking this chronic cycle isparamount to blocking disease progression. Excessive use of anti-VEGFreagents can also lead to the degeneration of capillary beds,particularly capillaries that have fenestrations like the ones found inthe ciliary body (Ford, K. M., Invest. Ophthalmol. Vis. Sci.53:7520-7527, 2012). The VEGF blockade in anti-cancer therapies has beenfound to damage the capillaries of the kidney and thyroid function inpeople treated locally for brain tumors (supra).

Current AMD therapies (Avastin and Lucentis, for example) are usedprimarily to treat the symptoms of AMD to reduce neovascularisation. Forchronic diseases such as AMD that involve loss of RGCs, the inventorshave appreciated that a twofold strategy of treatment would be betterthan a single therapy. First, choroid inflammatory progression and RGCloss in the inner retina itself must be ameliorated. Second, choroidinflammation in adjacent structures, such as underlying choroidal tissueca be directly targeted.

Co-administration treatment with the combination of an anti-VEGF and anconnexin 43 modulator results in improved benefits to the patient oversingle-agent therapy. The benefits of coadminstration are less frequentdosing (resulting in increased patient compliance and tolerability), andfewer side effects (such as new geographic atrophy). While Lucentis isused herein, other anti-VEGF compounds or ocular treatments may be usedin the methods of this invention, for example, Avastin, Eylea(aflibercept), Complement Factor D (lampalizumab), anti-SIP (iSONEP,sonepcizumab), and those AMD treatment agents described above.

Coadministration treatment is achieved by administering ranibizumab(Lucentis) by intravitreal injection on the same day as and prior toconexin 43 modulator administration. The vials containing bevacizumabare maintained at 4° C., and shaken well for at least one minute beforeusing. The eye is washed and draped in usual sterile fashion. Topicalanesthesia is given and a speculum is placed for adequate exposure. Theinjection quadrant is chosen by the treating physician and the site forinjection measured at 3.0 to 4.0 mm posterior to the limbus. A 28- or30-gauge needle is used to administer a 50 μL injection (0.5 mg) at 10mg/ml concentration of the bevacizumab formulation. After injection, aparacentesis is performed at the treating physician's discretion and thespeculum is removed. The dosings are applied once a month.

Next, the formulation comprising the connexin 43 modulator isadministered via intravitreal or intraocular injection or by othermethods of administration described herein. A 28- or 30-gauge needle isused to administer a 50 μL injection of the connexin 43 modulatorformulation. The connexin 43 formulation is the SEQ/Pept5 (“Peptide 5”)(SEQ ID NO: 168), dissolved in delivery medium at a concentration of 10mg/ml and delivered 0.5 mg (50 ul). Reflux of material duringadministration can be minimized or prevented by using a slow steady rateof administration and by application of gentle pressure with a counterpressure device (CPD) during administration of material and withdrawalof the cannula. The dosings are applied once a month (on the same day asthe Lucentis administration). The dose can also be 0.1 ug/ml, 0.5 ug/ml,1 ug/ml, 5 ug/ml, 2.5 ug/ml, 10 ug/ml, 50 ug/ml, 100 ug/ml, 1 mg/ml, 2.5mg/ml, 5 mg/ml, 10 mg/ml, 25 mg/ml, 50 mg/ml, 100 mg/ml, 250 mg/ml, or500 mg/ml. The delivery medium can also be 1×PBS, citrate, ascorbate, orborate based buffer, with 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% NaClconcentration for isotonicity. The pH can also be 3, 3.5, 4, 5, 6, 6.5,7, 7.4, or 8.

The patients are monitored by optical coherence tomography and visualacuity criteria to determine the effect of the coadministrationtreatment. The patient cohort treated with the co-administrationtreatments is compared to a patient group treated with Lucentis only,with the patient cohort treated with the co-administration exhibiting agreater visual acuity than the patient cohort treated with Lucentisonly. The patient cohort treated with the co-administration protocolexhibits a recovery in the choiroid, as monitored by optical coherencetomography.

Example 19: Formulation of Connexin or Pannexin Modulators with RhoKinase Inhibitors

This invention provides a formulation containing one or more agents thatenhance the ophthalmic properties of the rho kinase inhibitor compoundand connexin or pannexin modulator for separate or common administrationformulated in an aqueous medium whose pH is adjusted to enhance ocularsurface residence time and the bioavailability in the aqueous humor ofthe anterior chamber, and to reduce systemic exposure. The inventionprovides an aqueous formulation of the rho kinase inhibitor compound andconnexin or pannexin modulator that is suitable for therapeutic use andremains stable under normal use storage conditions for an extendedperiod of time. The formulation is useful for lowering intraocularpressure in mammals. For topical administration, one to two drops ofthese formulations can be delivered to the surface of the eye one tofour times per day. The aqueous ophthalmic formulations of thisinvention can have an increased residence time on the ocular surfaceand/or aqueous humor concentrations without a concomitant increase insystemic concentrations.

For the separate or common administration of the rho kinase inhibitorcompound and connexin or pannexin modulator, an aqueous pharmaceuticalformulation can comprise 0.001-2% rho kinase inhibitor compound,1.0-50,000 μM connexin or pannexin modulator, 1-100 mM buffer suitableto maintain the pH about 6.3-7.8, 0.01-2% surfactant, and a tonicityagent to maintain a tonicity about 200-360 mOsm/kg; “about” as usedherein, refers to ±15%. The pH is about 6.3-7.5, and preferably the pHis about 6.3-7.3. The compositions can further comprise, for example,sodium chloride, potassium chloride, calcium chloride and/or magnesiumchloride.

For the separate or common administration of the rho kinase inhibitorcompound and connexin or pannexin modulator, the concentration of therho kinase inhibitor in the aqueous formulation is in general 0.001-2%,preferably 0.01-0.5%, more preferably 0.01-0.4%, more preferably0.03-0.2%, and more preferably 0.03-0.15, or 0.03-0.1% (w/v). Bufferssuitable to maintain the pH between 6.3 and 7.8 include citrate,phosphate, maleate, or combination thereof. Suitable bufferconcentration is 1-100 mM, preferably 5-50 mM, more preferably 5-25 mM,and most preferably 10-20 mM.

For the separate or common administration of the rho kinase inhibitorcompound and connexin modulator, wherein the connexin modulator is aconnexin 43 modulator, the connexin 43 modulator may be present in theformulation at about 8 μM to about 20 μM final concentration, andalternatively the connexin 43 modulator is present at about 10 μM toabout 20 μM final concentration, or at about 10 to about 15 μM finalconcentration. In certain other embodiments, the connexin 43 modulatoris present at about 10 μM final concentration. In yet anotherembodiment, the connexin 43 modulator is present at about 1-15 μM finalconcentration. In other embodiments, the connexin 43 modulator ispresent at about a 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90μM, 100 μM, 10-200 μM, 200-300 μM, 300-400 μM, 400-500 μM, 500-600 μM,600-700 μM, 700-800 μM, 800-900 μM, 900-1000 or 1000-1500 μM, or 1500μM-2000 μM, 2000 μM-3000 μM, 3000 μM-4000 μM, 4000 μM-5000 μM, 5000μM-6000 μM, 6000 μM-7000 μM, 7000 μM-8000 μM, 8000 μM-9000 μM, 9000μM-10,000 μM, 10,000 μM-11,000 μM, 11,000 μM-12,000 μM, 12,000 μM-13,000μM, 13,000 μM-14,000 μM, 14,000 μM-15,000 μM, 15,000 μM-20,000 μM,20,000 μM-30,000 μM, 30,000 μM-50,000 μM, or greater, or any range orsubrange between any two of the recited doses, or any dose fallingwithin the range of from about 20 μM to about 50,000 μM.

For the separate or common administration of the rho kinase inhibitorcompound and connexin modulator, wherein the connexin modulator is aconnexin 43 modulator, the connexin modulator is any of the peptide orpolynucleotide sequences described above. In some aspects, the connexinmodulator can be the polynucleotide SEQ ID NO: 1. In some aspects, therho kinase inhibitor compound can be the amino isoquinolyl amideRhopressa.

Still other dosage levels between about 1 nanogram (ng)/kg and about 1mg/kg body weight per day of each of the connexin or pannexin modulatorsin the separate or common coadministration with the rho kinase inhibitorare described herein. In certain embodiments, the dosage of each of thesubject compounds will generally be in the range of about 1 ng to about1 microgram per kg body weight, about 1 ng to about 0.1 microgram per kgbody weight, about 1 ng to about 10 ng per kg body weight, about 10 ngto about 0.1 microgram per kg body weight, about 0.1 microgram to about1 microgram per kg body weight, about 20 ng to about 100 ng per kg bodyweight, about 0.001 mg to about 0.01 mg per kg body weight, about 0.01mg to about 0.1 mg per kg body weight, or about 0.1 mg to about 1 mg perkg body weight. In certain embodiments, the dosage of each of thesubject compounds will generally be in the range of about 0.001 mg toabout 0.01 mg per kg body weight, about 0.01 mg to about 0.1 mg per kgbody weight, about 0.1 mg to about 1 mg per kg body weight. If more thanone connexin or pannexin modulator is used, the dosage of eachanti-connexin agent need not be in the same range as the other. Forexample, the dosage of one connexin or pannexin modulator may be betweenabout 0.01 mg to about 10 mg per kg body weight, and the dosage ofanother connexin or pannexin modulator may be between about 0.1 mg toabout 1 mg per kg body weight, 0.1 to about 10, 0.1 to about 20, 0.1 toabout 30, 0.1 to about 40, or between about 0.1 to about 50 mg per kgbody weight. The dosage may also be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0,12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0,24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0,36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0,48.0, 49.0, 50.0, 52.5, 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5,75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 90.0, 92.5, 95.0, 97.5, or about100.0 mg per kg body weight, or any range or subrange between any two ofthe recited doses, or any dose falling within the range of from about0.1 to about 100 mg per kg body weight.

For the separate or common administration of the rho kinase inhibitorcompound and connexin or pannexin modulator, in one embodiment, thecombined use of the rho kinase inhibitor compound and connexin orpannexin modulator reduces the effective dose of any such agent comparedto the effective dose when said agent administered alone. In certainembodiments, the effective dose of the agent when used in combination isabout 1/15 to about ½, about 1/10 to about ⅓, about ⅛ to about ⅙, about⅕, about ¼, about ⅓ or about ½ the dose of the agent when used alone. Inanother preferred embodiment, the combined use of the rho kinaseinhibitor compound and connexin or pannexin modulator reduces thefrequency in which said agent is administered compared to the frequencywhen said agent is administered alone. Thus, these combinations allowthe use of lower and/or fewer doses of each agent than previouslyrequired to achieve desired therapeutic goals.

In one embodiment, the dose of the rho kinase inhibitor compound andconnexin or pannexin modulator when coadministered as separate or commonadministrations may be 10, 100 or 1000 fold lower than any of therecited doses set forth herein.

Surfactants (surface active agents) or solubilizing agents suitable forthe present invention are those acceptable for use in ophthalmicpreparations. The surfactants can be ionic or non-ionic. Preferably,this surfactant is non-ionic. Useful surfactants include but are notlimited to polysorbate 80, polyoxyl stearates, tyloxapol,polyethoxylated castor oils, poloxamers, polaxamines, medium and longchain fatty acids and phospholipids. The concentration of the surfactantin the formulation is about 0.01-3%, preferably 0.01-2%, more preferably0.1-1% w/v.

The tonicity agent is present in an amount to achieve a finalformulation tonicity between 220-360 mOsm/kG, preferably 250-340mOsm/kG, and most preferably between 260 and 320. The tonicity agent canbe ionic or non-ionic. Non-ionic tonicity agents include compoundscomprising 1,2-diols, such as glycerol, mannitol, erythritol; and sugarssuch as dextrose. Other non-ionic tonicity agents which also function ascosolvents can also be used such as polyethylene glycol and propyleneglycol. The non-ionic tonicity agent can be present in an amount of0-20%, preferably 0-10%, more preferably 0-5%. The non-ionic agents canbe selected from: glycerol, mannitol and dextrose, in an amount 2-6%.

The tonicity agent can also be ionic agents such as sodium chloride,potassium chloride, a balanced salt solution, sodium phosphate, orsodium citrate. The ionic tonicity agents can be present in an amount of0.3-1.5%, preferably 0.6-0.9%.

The surfactants, the tonicity agent, the buffer and any otheringredients introduced into the formulation preferably have goodsolubility in water, and have compatibility with other components in theformulation. Health regulations in various countries require thatmulti-dose ophthalmic preparations shall include a preservative. In oneembodiment, benzalkonium chloride is employed as a safe preservative;benzalkonium chloride may be used with disodiumethylenediaminetetraacetic acid (EDTA), a chelating agent, to enhanceits antimicrobial activity. Other suitable preservatives include benzylalcohol, methyl parabens, propyl parabens, borate, chlorobutanol, andbenzethonium chlorides. Typically, such preservatives are employed at alevel of from 0.001-1%, preferably, 0.001-0.25%, and most preferably0.001-0.2%.

Optionally, the formulation can include a viscosity enhancer to increasethe residence time of the formulation on the ocular surface. As anon-limiting example, hydroxypropyl methyl cellulose, can be used as aviscosity enhancer for the present invention.

Example 20: Method of Treating Glaucoma with the Combination of theConnexin 43 Modulator SEQ ID NO: 1 and the Rho Kinase Inhibitor

The connexin 43 modulator used is the polynucleotide SEQ ID NO:1. Therho kinase inhibitor used is Rhopressa. The connexin 43 modulator SEQ IDNO: 1 is formulated as a nanoparticle delivery formulation as describedabove, at a concentration of 100 μM of connexin 43 modulator total insolution. The concentration of the rho kinase inhibitor in the aqueousformulation is 0.03-0.1% (w/v), and formulated as described above.

Adult male Wistar rats are modeled for glaucoma with a retinal ischemiareperfusion. Retinal ischemia is modeled in the left eye (120 mmHg for60 min), with the right eye as the control, and then the connexin 43modulator formulation (neat, and infused into particles as describedabove) is injected intravitreally immediately as solutions in 1×PBSdelivery agent. Within one hour, the rho kinase inhibitor is then addedvia the administration of eye drops (50 uL). After reperfusion, Evan'sblue dye is injected intraperitoneally 4 hr after reperfusion tovisualize vessel leak using confocal microscopy and optical coherencetomography. Retinal whole mounts are collected at 8 hr and 28 days forCxn43 quantification, and 28 and 90 days for gangliol cell (RGC)densities quantification. Other cohorts of rats are treated with onlythe connexin 43 modulator, and other cohorts of rates are treated withonly the rho kinase inhibitor, at the same concentration as used in thecombination coadministration.

The results will indicate the vessel leak after 4 hr post-ischemia issignificantly reduced in the retinal for the eyes treated with thecombination of the rho kinase inhibitor and the connexin 43 modulatorcompared to control. It will be surprisingly shown that the connexin 43modulator works in the presence of the rho kinase inhibitor. The resultswill also show the surprising reduction in vessel leak for eyes treatedwith the combination of the rho kinase inhibitor and the connexin 43modulator compared to any eye with just single treatment.

Spot counts are also measured for all cohort models. It will be shownthat spot counts were reduced moreso with the combination treatment thanany single treatment.

After 28 days post-ischemia, cross-sections of the tissues are observedby confocal microscopy and coherence tomography. The mean Cxn43 count ismeasured for Evan's Blue dye-stained samples. The results will show thatthe combination formulations will result in lower Cxn43 expression thanany single administration formulation.

Retinal ganglion cell count is monitored for the models. The resultswill surprisingly show greater RGC survival for the coadministrationtreatment over any single administration treatment and untreated eyes(ischemia only).

Example 21: Connexin 43 is Upregulated Following Hemorrhage in DiabeticHumans

Human retina samples were obtained from human donors. FIGS. 45 and 46shows two human diabetic retinopathy retina images taken near ahaemorrhage, labelled for Cxn43 (red) and DAPI (blue) with rods andcones showing up at the bottom of FIG. 45.

Upon injury, Cxn43 levels Cxn43 rise as a result of injury to an extenteven greater than in normals as in the skin (as diabetic rats heal moreslowly). In diabetic retinopathy this results in inflammatory regionswith high Cxn43 and blood vessel leakage.

There appears to be a thickening of the RGC astrocyte layer, a piling upof small vessels and high levels of Cxn43 associated with them andastrocytes. Diabetics have reduced Cxn43 and vessel loss but not inareas of haemorrhage and neovascularisation, as there appears to behigher Cxn43 levels at inflammatory sites. The inventors have made thesurprising discovery that Cxn43 regulation in diabetic retinopathy is aviable drug target.

Example 22. In Vitro Block of Cxn43 Hemichannel Docking with ParticleFormulations

The blocking ability of Cxn43 modulators formulated in nanoparticles ormicroparticles was analyzed in vitro to establish the ability of theformulations to modulate Cxn43 activity. The microparticle (MP) andnanoparticle (Np) formulations were prepared as described above.

In vitro results from blocking Cxn43 hemichannels are shown in FIG. 47.After 8 h of exposure to a Cxn43 MP solution, Cxn43 labelling in ARPE-19cells was reduced (FIG. 47B); however, levels returned to normal after24 h (FIG. 47C). However, cells treated with Cxn43 MP loaded Nps andMps, exhibited reduced Cxn43 labelling at the later time point comparedto the 8 h evaluation. There were no differences in Cxn43 labellingafter 8 h of incubation with Nps (FIG. 47D) and Mps (FIG. 47F), whileafter 24 h of exposure, both Nps (FIG. 47E) and Mps (FIG. 47G) groupsexhibited reduced Cxn43 labelling confirming the delayed release andthus activity of Cxn43 MP. Each group exhibited a similar cell densitycompared to the control, indicating that no cytotoxicity occurred due tohemichannel blocking at the concentrations used.

Microparticle (MP) formulations composed of natural amino acids mightdegrade upon prolonged cellular incubation, thereby losing theiractivity. The half-life of native Cxn43 MP in bovine vitreous is about 2h, predicting that Cxn43 MP should be completely degraded after 14 h ifunprotected. This explains why Cxn43 hemichannels were blocked withinthe first few hours of incubation with the simple Cxn43 MP solutionwhile there was no difference in the level of Cxn43 labelling in thesame treatment group after 24 h (FIG. 47C) compared to the control (FIG.47A). Native Cxn43 MP in solution is likely to have been completelydegraded at that time point and existing Cxn43 hemichannels would havebeen continuously replaced by new ones. On the contrary, there was aprogressive reduction in Cxn43 plaque expression for both Cxn43 MPloaded Nps and Mps groups at 24 h (FIGS. 47E and 47), although nodifference was apparent between the two groups. It seemed that bothparticles protected the Cxn43 MP from degradation, sustained theirrelease and therefore prolonged their activity, although no effect wasseen within 8 h possibly due to insufficient Cxn43 MP released at thattime point. However, after 24 h greater concentrations of Cxn43 MP wereavailable correlating with increased hemichannel blockage and reducedCxn43 gap junction plaque labelling.

Example 23. Treatment of Cxn43 Upregulation with Cxn43 ParticleFormulations as Observed by GFAP Labelling in Retinal Cross-Sections

The microparticle (MP) and nanoparticle (Np) formulations describedabove were used to modulate Cxn43 upregulation in animal models, asobserved by GFAP labelling in retinal cross-sections.

Qualitative changes in Cxn43 and GFAP expression in association withretinal capillaries were observed following retinal ischaemia (FIG. 48).Twenty-eight days after reperfusion, Cxn43 was significantly upregulatedand mainly co-localised with GFAP (FIG. 48B). There was markedastrocytosis in the ischaemic regions with astrocytic foot processeslosing their normal organised appearance. Intravitreal injection ofCxn43 MP loaded Nps resulted in significantly (p<0.01) reduced Cxn43upregulation present at 28 d (FIG. 48D). Native Cxn43 MP in solution andCxn43 MP loaded Mps at 28 d and 90 d exhibited similar results, whereCxn43 expression was reduced along with astrocytes extending theirprocesses and proliferating into the periaxonal area (FIG. 48C-F). Cxn43spot counts, correlating with astrocytosis, were performed (FIG. 48G)and revealed that retinal Cxn43 was also significantly above normallevels (p<0.01) at 28 d (80.7±7.9) after ischaemia-reperfusion comparedto uninjured retinas (8.9±1.3). Treatment with native Cxn43 MP insolution resulted in a significantly reduced Cxn43 spot count to21.7±5.2 at 28 d (p<0.01); but was still significantly increasedcompared to the control (p<0.05). However, Nps loaded with Cxn43 MPdisplayed the greatest effect with the Cxn43 spot count down to 10.4±7.5after 28 d, with no significant difference compared to the uninjuredcontrol (p>0.05), confirming their protective and sustained releaseproperties. Mps loaded with Cxn43 MP were also able to limit Cxn43expression seen after 28 d (15.4±3.2) and 90 d (19.9±1.4) compared tothe untreated ischaemic eyes (p<0.01), again confirming the availabilityof stable Cxn43 MP at this time point.

These results indicate that the microparticle (MP) and nanoparticle (Np)formulations can modulate Cxn43 upregulation in a sustained manner inischemia-induced models in mammals.

Example 24. Treatment of Cxn43 Upregulation with Cxn43 ParticleFormulations as Observed by RGC Loss in Retinal Wholemounts

The microparticle (MP) and nanoparticle (Np) formulations describedabove were used to modulate Cxn43 upregulation as observed by RGC lossin retinal wholemounts.

The normal distribution of retinal ganlgion cells (RGC) in flatmounts ofuninjured retinas is shown in FIG. 49A, where a high cell density andclearly outlined retinal vasculature were visible. An example of the RGCdegeneration pattern in untreated ischaemic retinas is shown in FIG.49B, with RGC distribution 28 d after ischaemia-reperfusionsignificantly reduced with almost complete loss of blood vesseldelineation and large patches devoid of RGC noted in many areas. Thisindicates that the retina responded to the ischaemic insult withneurodegeneration following increased hemichannel mediated vascularpermeability. Eyes treated with native Cxn43 MP in solution (FIG. 49C)and Nps-Cxn43 MP (FIG. 49D) exhibited fewer patches of RGC loss. FIGS.49E and F illustrate the RGC distribution at 28 d and 90 d in eyestreated with Mps-Cxn43 MP with some RGC loss apparent. A summary of theRGC density (number/mm2) is depicted in FIG. 49G. Followingischaemia-reperfusion, the RGC density was significantly reduced(p<0.01) at 28 d in animals without treatment (1605±100) compared touninjured controls (2283±139), with only 70% of cells surviving. Thedensity in the Nps-Cxn43 MP group (1964±194) was similar to that of theCxn43 MP in solution treatment, while RGC counts after Mps-Cxn43 MPtreatment (1670±148 at 28 d and 1727±221 at 90 d) exhibited a trendtowards RGC sparing but exhibited no significant change compared tountreated eyes.

Ischaemia-reperfusion injury led to a significant loss of RGC in retinasat 28 d compared to uninjured controls, with intravitreal injection ofCxn43 MP in solution (p<0.05) or Nps-Cxn43 MP (p<0.01) significantlyreducing RGC death compared to no treatment, correlating with reducedCxn43 labelling. Although slower Cxn43 MP release from Mps alsodecreased Cxn43 levels at 28 d and 90 d following ischaemia-reperfusion,this treatment group failed to rescue RGC. This could be attributed toinsufficient initial Cxn43 MP release from Mps during the immediateacute stage after injury. In addition, Mps may get trapped in themeshwork due to their larger size compared to the Nps and released Cxn43MP would therefore have to diffuse larger distances to the retina,increasing the risk of enzymatic degradation. Results therefore suggestthat the RGC rescue effect may be more attributable to the initial burstof Cxn43 MP than the continuous slow release, suggesting that the soonerCxn43 hemichannels are blocked after ischaemia the better. Although wedo not have any Cxn43 and RGC count data from injured animals receivingno treatment at 90 d, previous studies have demonstrated that death ofRGC significantly increases with time, with more than 50% loss after 56d (S. S. L. Chew, et al., Invest. Ophthalmol. Vis. Sci. 52 (2011)3620-3629). Thus, although particle treatments did not show significantadvantages compared to the peptide in solution in terms of RGC survivalat the time points investigated, the sustained Cxn43 MP delivery effectmay still be beneficial for preservation of the RGC layer in the longterm.

Example 26. Alternative Method for Preparation of Nanoparticles andMicroparticles

An alternative route to the production of nanoparticles andmicroparticles is as follows: 100 mg of poly(lactic-co-glycolic acid)(PLGA) is placed in a test tube, followed by the addition of 1 ml ofdichloromethane solvent. Next, the tube is sealed and the polymerallowed to dissolve overnight. To a separate tube, a solution of 2 ml of0.3% w/w D-α-Tocopherol polyethylene glycol 1000 succinate (VitaminE-TPGS, Sigma 57668) emulsifier in water is prepared. The amount ofpolymer to add will determine the particle size.

For hydrophobic forms of the connexin 43 modulator: 5 μg of the connexin43 modulator is added in dry form to the PLGA solution. The tube isvortexed unti the solution is homogeneous by visual inspection.

For hydrophilic forms of the connexin 43 modulator: 50 ul of an aqueoussolution (at 10 uM concentration of the connexin 43 modulator in 1×PBSbuffer) is added to the PLGA solution. The solution is precooled byplacing the tube on ice for 30 seconds, then sonicated for 10 seconds toemulsify the connexin 43 modulator in the solution to achieve ahomogeneous opaque solution (VWR #97043-960). The tube is placed back onice.

For both forms of the connexin 43 modulator, the Vitamin E-TPGS tube isvortexed at high vortex speed using a benchtop vortexer (VWR Scientific#58816-121) while adding 1 ml of the PLGA-modulator solution dropwise.Vortexing is continued for 15 seconds.

Ice-water is placed in the sonicator. Next, the emulsion is sonicatedfor three 10 second bursts (40% amplitude for a 700 W sonicator). Thetube is moved around in the solution so as to ensure even sonication.Each sonication is performed 5 seconds apart to allow the solution tocool.

Next, 1 ml of 0.3% Vitamin E-TPGS is added in water to the emulsion viaglass pipette. Next, the emulsion is transferred dropwise into 45 ml of0.3% w/v Vitamin E-TPGS magnetically stirred at 360 rpm. The solution isstirred for 3 hours.

Next, the resulting solution is transferred into centrifuge tubes andcentrifuged in a fixed-angle rotor centrifuge for 15 min at 17,000×g.The supernatant is discarded. Next, 15 ml diH2O is added and the tube isvortexed to completely resuspend the particles. These steps are repeatedfour times. The fluid volume of the last pellet resuspension is 4-5 ml.A weight ratio of 1:2 trehalose:polymer is added (however, a smallaliquot of particles are prepared without trehalose for SEM imaging).

The nanoparticles are then transferred to a preweighed 5 ml centrifugetube and frozen at −80° C. for a minimum of 30 min. The nanoparticlesare then lyophilized 72 hr for a 5 ml volume and then stored at −80° C.

The inventions described and claimed herein have many attributes andembodiments including, but not limited to, those set forth or describedor referenced in this Detailed Disclosure. It is not intended to beall-inclusive and the inventions described and claimed herein are notlimited to or by the features or embodiments identified in this DetailedDisclosure, which is included for purposes of illustration only and notrestriction. A person having ordinary skill in the art will readilyrecognise that many of the components and parameters may be varied ormodified to a certain extent or substituted for known equivalentswithout departing from the scope of the invention. It should beappreciated that such modifications and equivalents are hereinincorporated as if individually set forth. The invention also includesall of the steps, features, compositions and compounds referred to orindicated in this specification, individually or collectively, and anyand all combinations of any two or more of said steps or features.

All patents, publications, scientific articles, web sites, and otherdocuments and materials referenced or mentioned herein are indicative ofthe levels of skill of those skilled in the art to which the inventionpertains, and each such referenced document and material is herebyincorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such patents, publications, scientific articles,web sites, electronically available information, and other referencedmaterials or documents. Reference to any applications, patents andpublications in this specification is not, and should not be taken as,an acknowledgment or any form of suggestion that they constitute validprior art or form part of the common general knowledge in any country inthe world.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,any of the terms “comprising”, “consisting essentially of”, and“consisting of” may be replaced with either of the other two terms inthe specification. Also, the terms “comprising”, “including”,containing”, etc. are to be read expansively and without limitation. Themethods and processes illustratively described herein suitably may bepracticed in differing orders of steps, and that they are notnecessarily restricted to the orders of steps indicated herein or in theclaims. It is also that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Under no circumstances may thepatent be interpreted to be limited to the specific examples orembodiments or methods specifically disclosed herein. Under nocircumstances may the patent be interpreted to be limited by anystatement made by any Examiner or any other official or employee of thePatent and Trademark Office unless such statement is specifically andwithout qualification or reservation expressly adopted in a responsivewriting by Applicants. Furthermore, titles, headings, or the like areprovided to enhance the reader's comprehension of this document, andshould not be read as limiting the scope of the present invention. Anyexamples of aspects, embodiments or components of the invention referredto herein are to be considered non-limiting.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

What is claimed is: 1-73. (canceled)
 74. A method for treating a subjectfor an ocular chronic wound, comprising topically administering to theeye of said subject a composition comprising an pharmaceuticallyacceptable carrier and from about 0.6 to about 60 mg/mL of a connexin 43antisense oligonucleotide having the sequence 5′ GTA ATT GCG GCA AGA AGAATT GTT TCT GTC 3′ (SEQ ID NO: 1).
 75. The method according to claim 74,wherein the ocular chronic wound is an ocular ulcer.
 76. The methodaccording to claim 74, wherein the ocular chronic wound is an ocularpersistent epithelial defect.
 77. The method according to claim 74,wherein the ocular chronic wound is a corneal persistent epithelialdefect.
 78. The method according to claim 74, wherein the connexin 43antisense oligonucleotide is an unmodified oligonucleotide.
 79. Themethod according to claim 74, wherein the connexin 43 antisenseoligonucleotide is a modified oligonucleotide.
 80. The method accordingto claim 79, wherein one or more nucleotide linkages and/or one or morenucleotide sugar moieties in the connexin 43 antisense oligonucleotideare modified.
 81. The method according to claim 74, wherein thecomposition contains about 0.6 mg/mL of said connexin 43 antisenseoligonucleotide.
 82. The method according to claim 74, wherein thecomposition comprises a reverse-thermosetting gel.
 83. The methodaccording to claim 82, wherein the reverse-thermosetting gel is anonionic polyoxyethylene-polyoxypropylene copolymer gel.
 84. The methodaccording to claim 83, wherein the nonionicpolyoxyethylene-polyoxypropylene copolymer is a pluronic gel.
 85. Themethod according to claim 84, wherein the pluronic gel comprisespoloxamer
 407. 86. The method according to claim 82, wherein thecomposition further comprises a buffer.
 87. The method according toclaim 74, wherein the composition comprises a transfection agent. 88.The method according to claim 74, wherein the composition comprises asurfactant.
 89. The method according to claim 74, wherein thecomposition comprises a preservative.
 90. The method according to claim74, wherein the composition comprises a salt.
 91. The method accordingto claim 74, wherein the composition comprises a compound selected fromthe group consisting of propylene glycol, dimethyl isosorbide, phosphatebuffered saline, water, isotonic water, deionized water, amonofunctional alcohol, a symmetrical alcohol, a carboxycellulose, acollagen, tropocollagen, a hyaluronic acid, a derived-hyaluronic acid, ahyaluronan and an oil.
 92. The method according to claim 74, wherein thecomposition comprises a compound selected from the group consisting ofhydroxymethylcellulose, hydroxyethylcellulose, carboxymethylcellulose,and hydroxypropylmethylcellulose.
 93. The method according to claim 74,wherein the composition is a slow or delayed release preparation. 94.The method according to claim 74, wherein the composition is formulatedfor delivery under a contact lens.
 95. The method according to claim 74,wherein the composition is contained within, delivered by, or attachedto a contact lens placed on the eye of said subject.
 96. The methodaccording to claim 74, wherein the composition is delivered inconjunction with a contact lens that is placed on the eye of saidsubject.
 97. A kit including a pharmaceutical composition comprising anophthalmologically acceptable carrier and from about 0.6 to about 60mg/mL of a connexin 43 antisense oligonucleotide having the sequence 5′GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC 3′ (SEQ ID NO: 1) andinstructions for use in treating an ocular persistent epithelial defect.98. The kit according to claim 97, further comprising a contact lens.